System for detecting rare cells

ABSTRACT

This disclosure provides a system for detecting rare cells. The system includes a substrate, extensions extending outwardly from the substrate and arranged about a center axis of the substrate to define a channel enabling the fluid to move radially from the center axis to an outer edge of the substrate, and a functionalized graphene oxide disposed on the extension. This disclosure also provides a method for detecting rare cells using the system of this disclosure. The method includes the steps of introducing a sample of fluid containing the rare cells into the inlet of the system such that the sample of fluid flows radially from the inlet toward the outer edge of the substrate and capturing the rare cells as the rare cells interact with the functionalized graphene oxide disposed on the extensions.

CROSS REFERENCE TO RELATED APPLICATIONS

The subject patent application is a continuation-in-part of U.S. patentapplication Ser. No. 14/347,073, filed on Mar. 25, 2014, which is theNational Stage of International Patent Application No.PCT/US2012/058013, filed on Sep. 28, 2012, which claims priority to andall the advantages of U.S. Provisional Application Ser. No. 61/541,814,filed on Sep. 30, 2011. The contents of U.S. patent application Ser. No.14/347,073, International Patent Application No. PCT/US2012/058013 andU.S. Provisional Application Ser. No. 61/541,814 are incorporated hereinby reference in their entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to a system, a microfluidicdevice, and a method for detecting rare cells in a fluid.

DESCRIPTION OF THE RELATED ART

As is well appreciated in the art, there are myriad technologicalobstacles in the identification, enumeration, detection, capture, andisolation of rare cells. These technological obstacles tend to limit thequantitative evaluation of rare cells, for example, in early diagnosisof metastatic diseases and effective monitoring of therapeutic responsein patients.

Some rare cells, e.g. circulating tumor cells (CTCs) and/or viabletumor-derived epithelial cells, have been identified in peripheral bloodfrom cancer patients and are likely the origin of intractable metastaticdisease. CTCs, as just one type of rare cell, tend to be present in anamount of about 1 CTC per 1 billion blood cells and tend to circulate inperipheral blood of patients with metastatic cancer. Detection,isolation, and capture of CTCs represent a potential alternative toinvasive biopsies during diagnosis of disease. More specifically, theability to identify, isolate, propagate and molecularly characterize CTCsubpopulations could further the discovery of cancer stem cellbiomarkers, expand the understanding of the biology of metastasis, andimprove the therapeutic treatment of cancer patients and the ultimatetreatment outcome. Many current strategies for isolating CTCs arelimited to complex analytic approaches that are typically very low yieldand low purity and that could be improved relative to sensitivity andaccuracy.

Many existing technologies utilize devices through which blood flowsover and around large three-dimensional structures for capturing CTCs.These structures tend to be expensive to produce, tend to act asobstacles to the flow of blood thereby decreasing the efficiency of thedevices, and tend to lack sensitivity for the CTCs thereby causing thedevice to have a low cell capture efficiency. Accordingly, there remainsan opportunity to develop an improved system and method of detectingrare cells.

SUMMARY OF THE DISCLOSURE

One embodiment of the instant disclosure provides a system for detectingrare cells in a fluid. The system comprises a substrate having an outeredge and defining a center axis, a plurality of extensions extendingoutwardly from said substrate and arranged about the center axis of thesubstrate to define a channel enabling the fluid to move radially fromthe center axis toward the outer edge of the substrate, and afunctionalized graphene oxide disposed on each of the plurality ofextensions.

This disclosure also provides a microfluidic device for detecting rarecells in a fluid. The microfluidic device comprises a silicon substratehaving an outer edge and defining a center axis, a plurality of metalextensions extending outwardly from the silicon substrate and arrangedabout the center axis of the silicon substrate to define a channelenabling the fluid to move radially from the center axis toward theouter edge of the silicon substrate, and a functionalized graphene oxidedisposed on each of the plurality of metal extensions.

This disclosure also provides a method for detecting rare cells usingthe system of this disclosure. The method includes the steps ofintroducing a sample of fluid containing the rare cells into the inletof the system such that the sample of fluid flows radially from theinlet toward the outer edge of the substrate and capturing the rarecells as the rare cells interact with the functionalized graphene oxidedisposed on the plurality of extensions.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Other advantages of the present disclosure will be readily appreciated,as the present disclosure becomes better understood by reference to thefollowing detailed description when considered in connection with theaccompanying drawings. It is to be understood that the drawings areillustrative and may not necessarily be drawn to scale.

FIG. 1A is a side view of a non-limiting embodiment of a microfluidicdevice and includes an inset magnified view of an extension (e.g. a goldnanopost), functionalized graphene oxide disposed on the extension,NeutrAvidin bound to the functionalized graphene oxide, a cancer cellantibody bound to the NeutrAvidin, and a cancer cell bound to theantibody.

FIG. 1B is a side view of a second non-limiting embodiment of amicrofluidic device including a silicon substrate, a plurality ofextensions (e.g. gold nanoposts) disposed on the silicon substrate,functionalized graphene oxide disposed on the plurality of extensions, aGMBS linker bound to the functionalized graphene oxide, NeutrAvidinbound to the GMBS linker, biotinylated EpCAM antibodies bound to theGMBS linker, and cancer cells bound to the antibodies.

FIG. 1C is an illustration of functionalized graphene oxide disposed ona plurality of extensions distributed in a leaf pattern on a substrate.

FIG. 1D is a magnified view of an extension (e.g. a gold nanopost) ofFIG. 1B including the functionalized graphene oxide disposed on the goldnanopost, the GMBS linker bound to the graphene oxide, the NeutrAvidinbound to the GMBS linker, the biotinylated EpCAM antibody bound to theNeutrAvidin, and the cancer cell bound to the biotinylated EpCAMantibody.

FIG. 1E is a perspective view of an extension disposed on a substrate.

FIG. 2A is a perspective view of one embodiment of a microfluidicdevice.

FIG. 2B is a perspective view of another embodiment of a microfluidicdevice that includes a pattern of gold nanoposts having a diameter ofabout 20 μm.

FIG. 2C is a schematic of a third embodiment of a microfluidic deviceand includes an inset magnified view of an extension (e.g. a goldnanopost) including the functionalized graphene oxide disposed on theextension, NeutrAvidin bound to the functionalized graphene oxide, anantibody bound to the NeutrAvidin, and a tumor cell bound to theantibody.

FIG. 2D is a top view of another embodiment of the microfluidic deviceincluding a microfluidic channel and a plurality of extensions disposedwithin the microfluidic channel.

FIG. 2E is a top view of yet another embodiment of a microfluidic deviceincluding a microfluidic channel and a plurality of extensions disposedwithin the microfluidic channel.

FIG. 2F is a magnified view of one embodiment of a pattern of aplurality of extensions, e.g. the extensions of FIGS. 2D and/or 2E,disposed on a substrate and illustrates a width (W₁) of an extension.

FIG. 2G is a top view of one embodiment of a microfluidic deviceincluding a magnified portion of a plurality of extensions distributedin leaf patterns on a substrate and rare cells disposed thereon.

FIG. 3 is a top view still another embodiment of the microfluidic deviceincluding a plurality of extensions disposed on a substrate.

FIG. 4A is a photograph of a circular pattern of extensions (e.g. goldnanoposts) of one embodiment wherein the extensions are disposed on asubstrate and wherein each extension has a diameter (e.g. W₁) of about20 μm.

FIG. 4B is a photograph of a flower pattern of extensions (e.g. goldnanoposts) of one embodiment wherein the extensions are disposed on asubstrate and wherein the unit length is about 100 μm.

FIG. 4C is a schematic of an extension (e.g. a gold nanopost) of oneembodiment wherein the extension is disposed on a substrate in a leafpattern.

FIG. 4D is a schematic of one embodiment of a design of a microfluidicdevice and includes an inset magnified view of a portion of a substratethat includes a plurality of extensions (e.g. gold nanoposts) disposedthereon in a flower pattern similar to the pattern set forth in FIG. 4B.

FIG. 5 is a schematic diagram of a series of method steps, one or moreof which may be utilized to form graphene oxide.

FIG. 6A illustrates simulation results of velocity fields andstreamlines of leaf patterns at 10 μm height of a simulated fluidchannel/chamber of a microfluidic device.

FIG. 6B illustrates simulation results of velocity fields andstreamlines of leaf patterns at 20 μm height of a simulated fluidchannel/chamber of a microfluidic device.

FIG. 6C illustrates simulation results of velocity fields andstreamlines of leaf patterns at 30 μm height of a simulated fluidchannel/chamber of a microfluidic device.

FIG. 7A illustrates simulation results of velocity fields andstreamlines of circular patterns at 10 μm height of a simulated fluidchannel/chamber of a microfluidic device.

FIG. 7B illustrates simulation results of velocity fields andstreamlines of circular patterns at 20 μm height of a simulated fluidchannel/chamber of a microfluidic device.

FIG. 7C illustrates simulation results of velocity fields andstreamlines of circular patterns at 30 μm height of a simulated fluidchannel/chamber of a microfluidic device.

FIG. 8 includes an SEM image of extensions disposed on a siliconsubstrate and an inset magnified view of one of the extensions.

FIG. 9A is an AFM image that illustrates one embodiment of a grapheneoxide sheet that has a thickness of about 2 nanometers.

FIG. 9B is a line graph that includes data from the AFM image of FIG. 9Aand further shows that one embodiment of a graphene oxide sheet has athickness of about 2 nanometers.

FIG. 9C is another AFM image that illustrates one embodiment of agraphene oxide sheet that has a thickness of about 3 nanometers.

FIG. 9D is a line graph that includes data from the AFM image of FIG. 9Cand further shows that one embodiment of a graphene oxide sheet has athickness of about 3 nanometers.

FIG. 10A is an SEM image of an extension (e.g. gold nanopost) that isnot washed with TBA Hydroxide and does not include any visible grapheneoxide sheets disposed thereon.

FIG. 10B is an SEM image of a second extension (e.g. gold nanopost) thatis not washed with TBA Hydroxide and does not include any visiblegraphene oxide sheets disposed thereon.

FIG. 10C is an SEM image of two extensions (e.g. gold nanoposts) thathave been washed with TBA Hydroxide and include a plurality of grapheneoxide sheets disposed thereon.

FIGS. 10D and 10E are SEM images of two additional extensions (e.g. goldnanoposts) that have been washed with TBA Hydroxide and include aplurality of graphene oxide sheets disposed thereon.

FIG. 11A is an SEM image of an extension (e.g. gold nanopost) includinga plurality of graphene oxide sheets disposed thereon and not washedwith IPA and DI water.

FIG. 11B is an SEM image of an extension (e.g. gold nanopost) includinga plurality of graphene oxide sheets disposed thereon and washed withIPA and DI water as described in the Examples.

FIG. 11C is an SEM image of self-assembled graphene oxide molecules on aplurality of extensions (e.g. gold nanoposts) disposed on a substrate inmultiple independent leaf patterns.

FIG. 12A is a fluorescence microscopy image of fluorescently labeledNeutrAvidin self-assembled on a plurality of extensions (e.g. goldnanoposts) that include functionalized graphene oxide disposed thereon.

FIG. 12B is a control fluorescence microscopy image of fluorescentlylabeled NeutrAvidin self-assembled on a plurality of extensions (e.g.gold nanoposts) that do not include functionalized graphene oxidedisposed thereon.

FIG. 13 A is a fluorescence microscope image of captured MCF-7 cancercells attached to antibodies disposed on a plurality of extensions (e.g.gold posts) having functionalized graphene oxide disposed thereon and ina microfluidic device similar to that illustrated in FIG. 2A.

FIG. 13B is another fluorescence microscope image of captured MCF-7cancer cells attached to antibodies disposed on a plurality ofextensions (e.g. gold posts) having functionalized graphene oxidedisposed thereon and in a microfluidic device similar to thatillustrated in FIG. 2A.

FIG. 13C is the fluorescence microscope image of FIG. 13B including amagnified portion thereof.

FIG. 13D is a fluorescence microscope image of captured MCF-7 cancercells attached to antibodies disposed on a plurality of extensions (e.g.gold posts) having functionalized graphene oxide disposed thereon and ina microfluidic device similar to that illustrated in FIG. 2G.

FIG. 13E is a second fluorescence microscope image of captured MCF-7cancer cells attached to antibodies disposed on a plurality ofextensions (e.g. gold posts) having functionalized graphene oxidedisposed thereon and in a microfluidic device similar to thatillustrated in FIG. 2G.

FIG. 13F is a third microscope image of captured MCF-7 cancer cellsattached to antibodies disposed on a plurality of extensions (e.g. goldposts) having functionalized graphene oxide disposed thereon and in amicrofluidic device similar to that illustrated in FIG. 2G.

FIG. 13G is a fourth microscope image of captured MCF-7 cancer cellsattached to antibodies disposed on a plurality of extensions (e.g. goldposts) having functionalized graphene oxide disposed thereon and in amicrofluidic device similar to that illustrated in FIG. 2G.

FIG. 14A is a fluorescence microscopy image of the extensions of acontrol microfluidic device described in the Examples wherein, at anexposure time of 1 second, no fluorescently labeled NeutrAvidin is seen.

FIG. 14B is a fluorescence microscopy image of the extensions of acontrol microfluidic device described in the Examples wherein, at anexposure time of 200 ms, no fluorescently labeled NeutrAvidin is seen.

FIG. 15A is an FT-IR spectrum of a sample of graphene oxide used in theExamples overlaid with an FT-IR spectrum of a sample of thePEG-functionalized graphene oxide formed in the Examples.

FIG. 15B is a magnified view of the FT-IR spectra of FIG. 15A from 1000to 1800 cm⁻¹.

FIG. 16A is a schematic of various method steps included in oneembodiment of the instant disclosure.

FIG. 16B is a schematic of various method steps included in anotherembodiment of the instant disclosure.

FIG. 17A is a bar graph showing the capture efficiency of variousexamples utilizing a microfluidic device similar to that illustrated inFIG. 2G and varying amounts of MCF-cells (3-5 cells, 10-20 cells, 100cells) spiked into 1 mL of whole blood.

FIG. 17B is a series of fluorescence microscope images of MCF-7 cell andwhite blood cell stained with DAPI, Cytokeratin, and CD45. The mergedimage identifies CTCs/cancer cells. CTCs/cancer cells are positive forDAPI and Cytokeratin. White blood cells are positive for DAPI and CD45.

FIG. 17C is a magnified fluorescence microscope images of MCF-7 cell andwhite blood cell bound to a plurality of extensions disposed in a leafpattern.

FIG. 17D is an SEM image of a cluster of rare cells proliferating whilebound to a plurality of extensions.

FIG. 18 is a bar graph showing the capture efficiency of variousexamples utilizing a microfluidic device similar to that illustrated inFIG. 2G using 100-1000 MCF-7 cells in a buffer solution at a flow rateof 1 mL/hr.

FIG. 19 is a bar graph showing the capture efficiency of variousexamples utilizing a microfluidic device similar to that illustrated inFIG. 2G using at different flow rates of 1 mL/hr, 2 mL/hr, 3 mL/hr.

FIG. 20 is a bar graph showing the capture efficiency of variousexamples utilizing a microfluidic device similar to that illustrated inFIG. 2G using 1000 MCF-7 cells and PC-3 cells in a buffer solution tocompare different cell lines' capture efficiency.

FIG. 21 is a bar graph showing the capture efficiency of variousexamples utilizing a microfluidic device similar to that illustrated inFIG. 2G using 1000 MCF-7 cells in buffer solution to compare amicrofluidic device including graphene oxide to a comparative silicondevice that is free of graphene oxide, as described in the examples.

FIG. 22 is an additional SEM image of a rare cell bound to a pluralityof extensions disposed in a leaf pattern and includes a magnified viewof the rare cell.

FIG. 23 is a fluorescence microscope image of captured and 6-days-grownMCF-7 cells bound to a plurality of extensions.

FIG. 24 is a second fluorescence microscope image of captured and6-days-grown MCF-7 cells bound to a plurality of extensions to identifyproliferating cells.

FIG. 25A is a table that sets forth optional non-limiting values of (A)of various embodiments of the leaf pattern set forth in FIG. 4A.

FIG. 25B is a table that sets forth optional non-limiting values of (B)of various embodiments of the leaf pattern set forth in FIG. 4A.

FIG. 25C is a table that sets forth optional non-limiting values of (C)of various embodiments of the leaf pattern set forth in FIG. 4A.

FIG. 25D is a table that sets forth optional non-limiting values of (D)of various embodiments of the leaf pattern set forth in FIG. 4A.

FIG. 26 is a schematic exploded view of another non-limiting embodimentof a microfluidic device including a plurality of bean-shaped extensionsarranged about a center axis of a substrate and rare cells (e.g.,circulating tumor cells) and white blood cells captured by thebean-shaped extensions.

FIG. 27 is a schematic illustration of the microfluidic device of FIG.26 showing an inlet at the center of the substrate and outlet at anouter edge of the substrate, and further shows a fluid flow profile withvelocity decreasing (from red to blue) from the inlet toward theoutlets.

FIG. 28 includes a photograph of the microfluidic device of FIG. 26 withfluid (e.g., blood) flow into the inlet at the center of the substrateand out of the outlets at the outer edge of the substrate.

FIG. 29 includes an SEM image of a portion of the microfluidic device ofFIG. 28 showing a plurality of bean-shaped extensions.

FIG. 30 is a schematic illustration of the microfluidic device of FIG.26 showing the radial flow of fluid from the inlet at the center of thesubstrate toward the outlet(s) at the outer edge of the substrate, andshowing that velocity decreases as the fluid radially flows from theinlet toward the outlet(s).

FIG. 31A includes an SEM image of a H1650 cell captured on thebean-shaped extension or micro-post.

FIG. 31B includes an SEM image of a magnified H1650 cell captured on thebean-shaped extension or micro-post.

FIG. 32 is a schematic, perspective view of a bean-shaped extensiondisposed on a substrate.

FIG. 33 is a schematic, perspective view of a bean-shaped extension witha functionalized graphene oxide layer disposed on the substrate.

FIG. 34A is a velocity magnitude profile across circular extensions of amicrofluidic device showing fluid flowing around the extensions at aconstant rate.

FIG. 34B is a fluid flow profile across circular extensions of amicrofluidic device.

FIG. 34C is a velocity magnitude profile across bean-shaped extensionsof another microfluidic device showing fluid flow slowing down near theconcave surface of the bean-shaped extensions.

FIG. 34D is a fluid flow profile across the bean-shaped extensions ofthe microfluidic device.

FIG. 35 is a velocity magnitude profile across a single bean-shapedextension of the microfluidic device showing flow converging behind theextension.

FIG. 36 is a velocity magnitude profile across the bean-shapedextensions of the microfluidic device near the inlet of the device(bottom arc), where flow occurs from the bottom toward the top of theprofile.

FIG. 37 is a particle tracing plot around the bean-shaped extensionsnear the inlet of the microfluidic device demonstrating typicalstreamlines (blue) and capture of 15 μm rigid particles (red) uponencountering an extension, where flow occurs from the bottom toward thetop of the plot.

FIG. 38 is a bar graph showing the comparison of H1650 cell capture fromwhole blood between a microfluidic device including the bean-shapedextensions and radial flow and a microfluidic device including circularextensions and linear flow at 1 and 10 mL/hr normalized to themicrofluidic device with the bean-shaped extensions and radial flow at 1mL/hr.

FIG. 39 is a bar graph showing non-specific white blood cell capture forthe experiment producing the results shown in FIG. 38 represented as thenumber of white blood cells per mL of whole blood.

FIG. 40 is a bar graph showing the effect of flow rates on capture ofH1650 cells spiked into whole blood by the microfluidic device includingthe bean-shaped extensions (Oncobean Chip) and radial flow showingcapture yields at 1, 2.5, 5, and 10 mL/hr.

FIG. 41 is an immunofluorescence image of cells stained for nucleus withDAPI with CD45 as a white blood cell marker and cytokeratin (CK) 7/8that stains the H1650 cancer cells.

FIG. 42 is a bar graph showing the clinical performance of the OncobeanChip showing CTC recovery from patients with pancreatic (Pa), breast(Br), and lung (L) cancer at 1 and 10 mL/hr.

FIGS. 43A-D are additional immunofluorescence images of lung CTCsstained recovered at 10 mL/hr showing the nucleus (FIG. 43A), theabsence of white blood cells (FIG. 43B), the presence H1650 cancer cells(FIG. 43C), and a merged field with all channel staining (FIG. 43D).

FIGS. 44A-D are immunofluorescence images of a cluster of CTCs from alung cancer specimen, where the nucleus is shown in FIG. 44A, theabsence of white blood cells is shown in FIG. 44B, the presence of tumormarker CK7/8 is shown in FIG. 44C, and a merger of all colors is shownin FIG. 44D.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure provides a system (20), (120) for detecting rarecells (22). Most typically, the rare cells (22) are present in samplesof blood, e.g. anticoagulated whole blood. However, it is alsocontemplated that the rare cells (22) may be present in samples of otherbodily fluids that may be, include, consist essentially of, or consistof, but are not limited to, saliva, mucus, excretions, and the like. Theterminology “consist essentially of” describes an embodiment wherein thebodily fluid is not diluted with a diluent. In one embodiment, the rarecells (22) may be transmitted via breath, i.e., breathing, sneezing,coughing, and the like, such that the rare cells (22) may be, at leastfor a time, airborne and thus still be present in a bodily fluid forpurposes of this disclosure. The bodily fluid may be utilized withoutpre-dilution, pre-labeling, pre-fixation, centrifugation, lysis, or anyother processing steps.

Transporting fluids, such as buffers, which may be miscible orimmiscible with various samples of blood and/or bodily fluids, may alsobe employed. In various embodiments, samples of blood, bodily fluids,and the like, may be evaluated in volumes of about 50 μL to about 5 mL,about 100 μL to about 1 mL, or about 250 μL to about 550 μL. However,the present disclosure is not limited to these volumes or to dilution ofbodily fluids. In one embodiment, about 1 mL of sample is utilized. Inother embodiments, 1 to 20, 2 to 19, 3 to 18, 4 to 17, 5 to 16, 6 to 15,7 to 14, 8 to 13, 9 to 12, or 10 to 11, mL of sample are utilized. Anyof the aforementioned values may, for example, vary by 1, 2, 3, 4, 5,10, 15, 20, or 25+% in varying non-limiting embodiments. All values, andranges of values, between and including the aforementioned values arealso hereby expressly contemplated in various non-limiting embodiments.

The particular type of rare cells (22) contemplated in this disclosureis not limited. In one embodiment, the rare cells (22) are furtherdefined as circulating tumor cells (22) (CTCs), see e.g. FIG. 17B. Inother embodiments, the rare cells are chosen from the group ofendothelial cells, fetal cells, and/or cells of hemopoietic origin (e.g.platelets, sickle cell red blood cells, and subpopulations ofleukocytes). In still other embodiments, the terminology “rare cells”alternatively describes exosomes, microvessicles, bacteria, viruses,protists, and/or fungi.

The rare cells, such as CTCs, may be present, for example in blood,bodily fluids, and the like, in any amount, e.g. in amounts of from 0.01to 10, from 0.1 to 10, from 1 to 10, from 1 to 20, from 1 to 30, from 1to 40, from 1 to 50, from 1 to 60, from 1 to 70, from 1 to 80, from 1 to90, from 1 to 100, from 100 to 1000, from 200 to 900, from 300 to 800,from 400 to 700, from 500 to 600, from 1 to 5, or 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, rare cellsper one billion total blood cells. Alternatively, the rare cells may bepresent in amounts of greater than 0.01, 0.1, 1, 10, 50, 100, 500, 1000,5000, or 10000, rare cells per one billion total blood cells. Any of theaforementioned values may, for example, vary by 1, 2, 3, 4, 5, 10, 15,20, or 25+% in varying non-limiting embodiments. All values, and rangesof values, between and including the aforementioned values are alsohereby expressly contemplated in various non-limiting embodiments. Rarecells present in bodily fluids other than blood and/or CTCs may also bepresent in the aforementioned amounts. However, the instant disclosureis not limited to these amounts of rare cells present in bodily fluidsand it is contemplated that higher or lower amounts may also beutilized.

The system of this disclosure includes a substrate (24), (124), anextension (26), (126) coupled to the substrate (24), (124) and extendingoutwardly from the substrate (24), (124) and a functionalized grapheneoxide (28), (128) disposed on the extension (26), (126). Variousembodiments of the system (20) are set forth in and described below withreference to FIGS. 1A-D, 2A-G, 3, and 4A-D. Various embodiments of thesystem (120) are set forth in and described below with reference toFIGS. 26-33. Typically, as a bodily fluid flows over the substrate, e.g.through a microfluidic channel (32), (132) and/or a microfluidic chamber(56), (156), rare cells in the bodily fluid come into contact with thefunctionalized graphene oxide, one or more binding agents, markers,proteins, etc. and become immobilized, e.g. on the surface of theextension (26), (126) or as attached to one or more binding agents,markers, proteins, etc. Each of the substrate (24), (124), the extension(26), (126), and the functionalized graphene oxide (28), (128) isdescribed in greater detail below.

The System (20)

Substrate:

The substrate (24) is not particularly limited in this disclosure andmay be further defined as being, including, consisting essentially of,or consisting of, a metal, plastic, polymer, inorganic compound, glass,silicon (e.g. —Si—Si—), silicone (e.g. —Si—O—Si— or PDMS), epoxy,semiconductors, and/or combinations thereof. The terminology “consistessentially of” typically describes that the substrate (24) includes oneor more of the particular aforementioned materials and is free of, orincludes less than 0.1 or 1, weight percent, of dissimilar materials.The substrate (24) may be fabricated using any technique known in theart including, but not limited to, molding, photolithography,electroforming, machining, chemical vapor deposition, and the like.

The substrate (24) may also be further defined as a device, layer, film,coating, sheet, skin, chip, block, or wafer. In various embodiments, thesubstrate (24) is further defined as a tri-layered substrate thatincludes a silicon layer, an SiO₂ layer, and a PDMS (i.e.,polydimethylsiloxane) layer, as set forth in FIG. 1A. Alternatively, thesubstrate (24) may be further defined as a single layer. In oneembodiment, additional layers, e.g. the SiO₂ layer and the PDMS layer,are disposed on the single layer and may be individually described asone or more supplemental (or support) layers (34). Depending on overalldesign and shape, one or more of the substrate (24) and/or one or moresupplemental layers (34) may be independently further defined as anoutermost layer, an innermost layer, or an interior layer, e.g. of adevice or of the system (20). In other embodiments, the substrate (24)and/or one or more supplemental layer (34) may be, include, consistessentially of, or consist of, one or more of polyethylene terephthalate(PET), polyimide, polyether ether ketone (PEEK), and/or combinationsthereof.

The substrate (24) and/or the one or more supplemental layers (34) maybe bonded together by any means known in the art including use ofadhesives, chemical bonding techniques, and physical bonding techniques.In one embodiment, the substrate (24) includes an SiO₂ layer that isbonded to the substrate (24) using oxygen plasma treatment.

The substrate (24) and one or more supplemental layers (34) are notlimited to any particular configuration or structure and each of thesubstrate (24) and one or more supplemental layers (34) mayindependently be disposed in any order or configuration relative to oneanother. All combinations of these layers and configurations are hereinexpressly contemplated. Each of the substrate (24) and supplementallayers (34) are also not particularly limited to any particularcross-section and each may independently have, but is not limited tohaving, a rectangular cross-section, a square cross-section, atriangular cross-section, a circular or oval cross-section, an“P”-shaped cross-section, a “C”-shaped cross-section, an “L”-shapedcross-section, a “T”-shaped cross-section, a “U”-shaped cross-section,or a “W” shaped cross-section. The substrate (24) and supplementallayers (34) may be solid, hollow, or have solid sections and hollowsections.

The overall size of each of the substrate (24) and supplemental layers(34) is not particularly limited. In one embodiment, the substrate (24)has dimensions of about 35 mm×10 mm×3 mm. However, these dimensions arenot limiting and may vary. Suitable non-limiting examples of substrates(24) and supplemental layers (34) have length, width, and heightdimensions on the scale of 1 to 100, 1 to 75, 1 to 50, 1 to 25, 1 to 20,1 to 15, 1 to 10, 1 to 5, or 0.1 to 1, inches, centimeters, and/ormillimeters. Any of the aforementioned values may, for example, vary by1, 2, 3, 4, 5, 10, 15, 20, or 25+% in varying non-limiting embodiments.All values, and ranges of values, between and including theaforementioned values are also hereby expressly contemplated in variousnon-limiting embodiments. It is also contemplated that a microfluidicdevice, as described in greater detail below, may have the same ordifferent dimensions from one or more of the substrate (24) and/or thesupplemental layer(s) (34).

Extension:

The system also includes the extension (26) coupled to the substrate(24), and extending outwardly from the substrate (24), as set forth inFIGS. 1-4. The terminology “extension” may describe a single extension,two extensions, or a plurality of extensions, in various embodiments,throughout. Said differently, whenever the terminology “extension” isused, that terminology may describe various embodiments including asingle extension, two extensions, or a plurality of extensions.

The extension (26) may extend outwardly from the substrate (24)approximately perpendicularly to a longitudinal axis (L₁) or may extendoutwardly at another angle to the substrate (24) and/or the longitudinalaxis (L₁), e.g. at an obtuse or acute angle, such as 30°, 45°, or 60°.The extension (26) may be coupled to the substrate (24) via any meansknown in the art such as through chemical and physical connections, e.g.with adhesives, via chemical bonding, and the like. Similarly, theextension (26) may be coupled to the substrate (24) in direct contactwith the substrate (24) or in indirect contact with the substrate (24),e.g. separated by one or more layers, compounds, molecules, etc. As anadditional example, the extension (26) may be disposed in direct contactwith an intermediate or supplemental layer (34) or connection which, inturn, may be disposed either directly or indirectly with the substrate(24). It is contemplated that the extension (26) may still be coupled tothe substrate (24) even though there is no direct contact therebetween.

The extension (26) typically has an upper end (54) and a lower end (56)and a vertical axis (V) that extends through the upper and lower ends(54, 56), as shown in FIG. 1E. Typically, the upper and lower ends (54,56) extend along the vertical axis (V). The extension (26) alsotypically has a horizontal axis (H₁) that extends between the upper andlower ends (54, 56), as also shown in FIG. 1E.

The extension (26) may be disposed substantially perpendicularly to thesubstrate (24) and/or horizontal axis (H₁) or disposed transversely(i.e., at any angle) to the substrate (24) and/or horizontal axis (H₁).It is also contemplated that the extension (26) may be disposed suchthat the horizontal axis (H₁) is disposed approximately parallel to, ortransverse to, the substrate (24). The extension (26) may be furtherdefined as a post or rod, e.g. a micro-post, micro-rod, nanopost,nanorod, etc. In one embodiment, the extension (26) is further definedas an electrode. Typically, the extension (26) has micro- or nano-scaledimensions.

In various embodiments, the extension (26), e.g. a nanopost, has aheight (e.g. H₂) of about 100 nm and a width or radius of about 10 μm.In other embodiments, the extension (26) has a height (H₂) of about 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95nm, or ranges thereof. In still other embodiments, the extension (26)has a height (e.g. H₂) of about 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 205, 210, 215,220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285,290, 295, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nm, or rangesthereof. In even further embodiments, the extension (26) has a height(e.g. H₂) of from 10 to 2000, from 50 to 2000, from 100 to 2000, from200 to 1900, from 300 to 1800, from 400 to 1700, from 500 to 1600, from600 to 1500, from 700 to 1400, from 800 to 1300, from 900 to 1200, orfrom 1000 to 1100, nm, or ranges thereof. In other embodiments, theextension (26) has a diameter or width (e.g. W₂) of from 100 nm to 1000micrometers or from 100 nm to 1000 nm, from 150 to 950, from 200 to 800,from 250 to 750, from 300 to 700, from 350 to 650, from 400 to 600, from450 to 550, or from 500 to 550, nm, or ranges thereof. In still otherembodiments, the extension (26) has a diameter or width (e.g. W₂) offrom 20 to 100, from 25 to 95, from 30 to 90, from 35 to 85, from 40 to80, from 45 to 75, from 50 to 70, from 55 to 65, or from 60 to 65, nm,or ranges thereof. Any of the aforementioned values may, for example,vary by 1, 2, 3, 4, 5, 10, 15, 20, or 25+% in varying non-limitingembodiments. All values, and ranges of values, between and including theaforementioned values are also hereby expressly contemplated in variousnon-limiting embodiments.

It is contemplated that, relative to the height/thickness of amicrofluidic device or channel (32) or chamber (56), described ingreater detail below, and e.g. as shown as T₁ in FIG. 2A or T₂ or T₃ inFIG. 2G, the extension (26) may appear essentially two dimensional, ascould be determined by one of skill in the art. For example, if theheight/thickness of a microfluidic device or channel (32) or chamber(56) is 40-50 micrometers, even a 500 nanometer height of an extension(26) is only 1% of the height/thickness of the microfluidic device orchannel (32) or chamber (56). In a similar scenario, a 5 nanometerheight of an extension (26) is only 0.01% of the height/thickness of amicrofluidic device, channel (32), or chamber (56). Moreover, a 1nanometer height of an extension (26) is only 0.002% of theheight/thickness of a microfluidic device, channel (32), or chamber(56). In similar embodiments, the height of the extension (26) is small,as appreciated by a person of skill in the art, compared to theheight/thickness of the microfluidic device, channel (32), or chamber(56), that the extension (26) appears to be almost two-dimensional.Similarly, even under light microscopy and modest magnification (e.g.50-500×), the height of the extension (26) may appear essentiallytwo-dimensional when compared to the height/thickness of themicrofluidic device, channel (32), or chamber (56), as appreciated by aperson of skill in the art.

The extension (26) may be, include, consist essentially of, or consistof, a plastic, polymer (such as polymethylmethacrylate (PMMA)) or metalor combinations thereof. In one embodiment, the metal is gold (e.g. theextension (26) may be formed from gold). Alternatively, the metal maybe, include, consist essentially of, consist of, or be chosen from thegroup of, transition metals, precious metals, rare earth metals, andcombinations thereof. In various embodiments, it is contemplated thatthe extension (26) be, include, consist of, or consist essentially of, ametal, such as gold, silver, and/or copper, and/or a mixed metalcompound such as indium-tin oxide (ITO). The terminology “consistessentially of” typically describes that the extension (26) includes oneor more of the aforementioned materials and is free of, or includes lessthan 0.1 or 1, weight percent, of a non-metal or a non-mixed metalcompound or another of the aforementioned materials.

The extension (26) may be formed by any method known in the art. In oneembodiment, the extension (26) is formed by evaporating and patterningmetal layers, e.g. Cr/Au layers (10/100 nm). In various embodiments, theextension (26) can be formed using a lift-off process which typicallyallows for fine patterns to be formed. A photoresist may be coated on asilicon substrate (24) and patterned by photolithography, see e.g. FIG.5. Then metal layers may be deposited on the silicon wafer.Subsequently, the substrate (24) may be immersed in acetone or aphotoresist remover solution. A patterned gold layer typically remains.In other embodiments, a shadow mask can be used in conjunction withdepositing a layer, e.g. a gold layer. Electroplating techniques mayalso be utilized throughout this disclosure.

The extension (26) may be disposed on any one or more portions orsegments of the substrate (24), microfluidic device, channel (32),and/or chamber (56). In various embodiments, the extension (26) isdisposed on or in/within a microfluidic channel (32) or a microfluidicchamber (56), as first introduced above. In other embodiments, more thanone extension (26) is disposed on or in the substrate (24), microfluidicdevice, channel (32), and/or chamber (56) in a pattern, for example, asset forth in FIGS. 2F, 3, and 11C. It is contemplated that a totalnumber of extensions (26) may exceed hundreds, thousands, hundreds ofthousands, millions, tens of millions, etc.

The total number of extensions (26) is not particularly limited. Theextension (26) itself may be formed in a shape/pattern and/or aplurality of extensions (26) may be, as a whole, set forth in ashape/pattern that may be the same or different than the pattern of anyindividual extension (26). Each individual extension (26) may have ashape/pattern that is the same or different from any one or more otherextensions (26). Similarly, the plurality of extensions may be segmentedinto one or more segments and each segment may individually have ashape/pattern than is the same or different from the shape/pattern ofany other segment and/or from any shape/pattern of any individualextension (26).

The size and geometry of these patterns is also not particularlylimited. In one embodiment, the diameter of a pattern for an individualextension (26) is about 20 micrometers. In another embodiment, the unitlength of a pattern for an individual extension (26) is about 100micrometers. As set forth in FIGS. 4A-4C, individual extensions (26) mayhave a circular, flower, or leaf shape/patterns However, these shapesand patterns are not particularly limiting. Any of the aforementionedshapes/patterns are not limited and each may individually be furtherdefined as a geometric shape/pattern, a non-geometric shape/pattern, auniform or non-uniform shape/pattern, or as a gradient shape/pattern.Alternatively, the aforementioned shape/pattern of any one or moreindependent extensions (26), segments, or plurality of extensions (26)may not have any defined shape or pattern and may be described as randomor amorphous. Still further, any of the aforementioned shapes/patternsmay be as described below relative to the shapes/patterns of themicrofluidic channel (32) and/or microfluidic chamber (56).

For example, in FIG. 4C, the dimensions of (A) may be 2 to 500 μm or anyother value or range of values set forth in the table of FIG. 25A or anyvalue or range of values therebetween. The dimensions of (B) may be 5 to2000 μm or any other value or range of values set forth in the table ofFIG. 25B or any value or range of values therebetween. The dimensions of(C) may be 2 to 1000 μm or any other value or range of values set forthin the table of FIG. 25C or any value or range of values therebetween.The dimensions of (D) may be 2 to 500 μm or any other value or range ofvalues set forth in the table of FIG. 25D or any value or range ofvalues therebetween. In one embodiment, the dimensions of (A), (B), (C),and (D), are 25 μm, 100 μm, 36.5 μm, and 25 μm, respectively. Any of theaforementioned values may, for example, vary by 1, 2, 3, 4, 5, 10, 15,20, or 25+% in varying non-limiting embodiments. All values, and rangesof values, between and including the aforementioned values and thevalues in the tables are also hereby expressly contemplated in variousnon-limiting embodiments.

In other embodiments, the extensions (26) are disposed in patterns, e.g.patterns having a length, width, and/or spacing of about 150 nanometers(e.g. with about a 1.5 micrometer pitch). In still other embodiments,the extensions (26) are disposed with a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, of100 μm, distance between extensions (26) and a shift between at leasttwo rows of an independent distance that may be one of the valuesdescribed immediately above, e.g. 10, 20, 30, 40, 50, 60, 70, 80, 90, or100 μm, or ranges thereof. In one embodiment, the extensions (26) aredisposed in an equilateral triangular arrangement with a 50 μm distancebetween extensions (26) and a 50 μm shift after every 3 rows. Any of theaforementioned values may, for example, vary by 1, 2, 3, 4, 5, 10, 15,20, or 25+% in varying non-limiting embodiments. All values, and rangesof values, between and including the aforementioned values are alsohereby expressly contemplated in various non-limiting embodiments.

In other embodiments, the pitch distances may be from 1 μm to 1000 μm(i.e., 1 mm) In various embodiments, the pitch distance is from 1 to100, 5 to 95, 10 to 90, 15 to 85, 20 to 80, 25 to 75, 30 to 70, 35 to65, 40 to 60, 45 to 55, or 50 to 55, μm. In other embodiments, the pitchdistance is from 100 to 1000, from 125 to 975, from 150 to 950, from 175to 925, from 200 to 900, from 225 to 875, from 250 to 850, from 275 to825, from 300 to 800, from 325 to 775, from 350 to 750, from 375 to 725,from 400 to 700, from 425 to 675, from 450 to 650, from 475 to 625, from500 to 600, from 525 to 575, or from 550 to 575, μm. Any of theaforementioned values may, for example, vary by 1, 2, 3, 4, 5, 10, 15,20, or 25+% in varying non-limiting embodiments. All values, and rangesof values, between and including the aforementioned values are alsohereby expressly contemplated in various non-limiting embodiments.

Functionalized Graphene Oxide:

The functionalized graphene oxide (28) of this disclosure is disposed onthe extension (26) or on more than one extension (26), for examples, asshown in FIG. 8. The graphene oxide (28), either pre- orpost-functionalization, may be disposed on, or attached to, one or moreextensions (26) by any means known in the art including both physicaland chemical attachment including covalent bonding, electrostaticattraction, etc. In one embodiment, the extensions (26) are exposed to acompound, such as TBA Hydroxide, that facilitates binding of thegraphene oxide (28) to the extensions (26), see e.g. FIGS. 10A-E.Without intending to be bound by any particular theory, it is believedthat TBA Hydroxide forms a cation (+) that interacts with anions (−)(e.g. gold anions) of the extensions (26).

The extensions (26) may include one or more types of (functionalized)graphene oxide (28) and one or more markers, binding agents, etc.,bonded or attached thereto, see e.g. FIGS. 16A/B. For example, multiplebinding agents may bind to the same or different cells and may be placedin the same or different microfluidic channels (32) or on the same ordifferent extensions (26).

Graphene oxide is a single layer form of graphite oxide and can befurther defined as a form of graphene that includes oxygen functionalgroups on basal planes and edges. Typically, graphene oxide is describedas a strong paper-like material. Graphene oxide, whether functionalizedor not functionalized, may have a thickness of about 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0nm, or up to 50 nm, e.g. in tenth- or half-nanometer increments, each±0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0, nm, see e.g. FIGS.9A-D. In various embodiments, the combined height of the(functionalized) graphene oxide and the extension is from 1 μm to 1000μm (i.e., 1 mm) In various embodiments, the combined height is from 1 μmto 100, 5 to 95, 10 to 90, 15 to 85, 20 to 80, 25 to 75, 30 to 70, 35 to65, 40 to 60, 45 to 55, or 50 to 55 μm. In other embodiments, thecombined height is from 100 to 1000, from 125 to 975, from 150 to 950,from 175 to 925, from 200 to 900, from 225 to 875, from 250 to 850, from275 to 825, from 300 to 800, from 325 to 775, from 350 to 750, from 375to 725, from 400 to 700, from 425 to 675, from 450 to 650, from 475 to625, from 500 to 600, from 525 to 575, or from 550 to 575 μm. Any of theaforementioned values may, for example, vary by 1, 2, 3, 4, 5, 10, 15,20, or 25+% in varying non-limiting embodiments. All values, and rangesof values, between and including the aforementioned values are alsohereby expressly contemplated in various non-limiting embodiments.

In one embodiment, the graphene oxide is formed from graphite, asdescribed in D. Li, M. B. Muller, S. Gilje, R. B. Kaner, and G. G.Wallace, “Processable aqueous dispersions of graphene nanosheets,”Nature Nanotechnology, vol. 3, pp. 101-105, 2008, which is expresslyincorporated herein by reference in a non-limiting embodiment. Inanother embodiment, the graphene oxide is formed using the procedure asdescribed in Z. Wei, D. E. Barlow, and P. E. Sheehan, “The Assembly ofSingle-Layer Graphene Oxide and Graphene Using Molecular Templates,”Nano Letters, Vol. 8, No. 10, pp. 3141-3145, 2008, also expresslyincorporated herein by reference in a non-limiting embodiment. In stillanother embodiment, the graphene oxide is formed from graphene sheetsthat are formed using the procedure as described in H. Wang, X. Wang, X.Li, H. Dai, “Chemical Self-Assembly of Graphene Sheets,” Nano Research,Vol. 2, pp. 336-342, 2009, also expressly incorporated herein byreference in a non-limiting embodiment. In even another embodiment, thegraphene oxide is formed using the procedure described in X. Sun, Z.Liu, K. Welsher, J. T. Robinson, A. Goodwin, S. Zaric, H. Dai“Nano-Graphene Oxide for Cellular Imagine and Drug Delivery” NanoResearch, Vol. 1, pp. 203-212, 2008, also expressly incorporated hereinby reference in a non-limiting embodiment. It is also contemplated thatthe graphene oxide may be formed using the procedure described in U.S.Pat. App. Pub. No. US 2010/0028681, which is also expressly incorporatedherein by reference in a non-limiting embodiment.

In one embodiment, graphene oxide sheets are formed byexfoliation-reintercalation-expansion methods, as described above. Inanother embodiment, ground natural graphite is intercalated by oleum inthe presence of sodium nitrate. The product may then be treated with anaqueous solution of tetrabutylammonium (TBA) hydroxide and suspended byPL-PEG-NH₂ in DMF.

In still other embodiments, the graphene oxide sheets can be formed onthe surface of the extensions (26). Without intending to be bound by anyparticular theory, it is believed that selective absorption of graphenesheets onto the extensions (26), e.g. gold patterns, can occur viaelectrostatic interactions. Suitable, but non-limiting, graphene oxide(28) sheets can have sizes, e.g. length and/or width, from about 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800,825, 850, 875, 900, 925, 950, or 975 nm, to about 1 micrometer.Alternatively, the upper range of the length and/or width may be up to100 micrometers, e.g. in increments of, e.g. half, tenth, or hundredths,or thousandths, of a micrometer. Any of the aforementioned values may,for example, vary by 1, 2, 3, 4, 5, 10, 15, 20, or 25+% in varyingnon-limiting embodiments. All values, and ranges of values, between andincluding the aforementioned values are also hereby expresslycontemplated in various non-limiting embodiments.

The graphene oxide (28) that is functionalized typically forms stablesuspensions in water and can aggregate in salt or other biologicalsolutions. In various embodiments, the graphene oxide (28) isfunctionalized with one or more functional groups including, but notlimited to, aliphatic groups, aromatic groups, nitrogen including groupssuch as amines and amides, carboxyl groups, sulfur including groups,phosphorous including groups, and the like. Alternatively, the grapheneoxide (28) can be functionalized with one or more markers, antibodies,antigens, proteins, tumor specific binding agents (e.g. anti-EpCAM), andthe like. In another embodiment, the terminology “tumor specific bindingagent” describes an agent that binds to a nonhemopoietic cell that canform a tumor, such as a cell not of hemopoietic origin, excluding bloodcells and immune cells, but including epithelial cells, endothelialcells, neurons, hepatocytes, nephrons, glial cells, muscle cells, skincells, adipocytes, fibroblasts, chondrocytes, osteocytes, andosteoblasts. The binding agent may bind to a cell surface marker that isspecific for a type of cell that can form a tumor and that is notnormally found in circulating blood. In an alternative, the bindingagent may bind to a cell surface marker that is specific for atransformed cell. Such agents may also bind to healthy cells circulatingin blood from non-pathogenic origins, e.g., venipuncture or trauma. Inother embodiments, Streptavidin and/or one or more antibodies forvarious viruses may be utilized.

In various embodiments, the graphene oxide (28) is functionalized withone or more markers that allows for identification, enumeration,detection, capture, and/or isolation of genomic DNA, cDNA, or mRNAsequences, proteins or other intracellular contents that are indicativeof a type or presence of a particular tumor, determination of thepresence or absence of certain mutations in EGFR, HER2, prostatespecific antigen TMPRSS2-ERG, CD133, CD44, CD24, epithelial-specificantigen (ESA), Nanog, 25 BMI1, and the like. Alternatively, the grapheneoxide (28) may be functionalized with one markers that allows foridentification, enumeration, detection, capture, and/or isolation ofcells related, but not limited, to one or more of the following cancers:ostate, lung, adenocarcinoma, adenoma, adrenal cancer, basal cellcarcinoma, bone cancer, brain cancer, breast cancer, bronchi cancer,cervical dysplasia, colon cancer, epidermoid carcinoma, Ewing's sarcoma,gallbladder cancer, gallstone tumor, giant cell tumor, glioblastomamultiforma, head cancer, hyperplasia, hyperplastic corneal nerve tumor,in situ carcinoma, intestinal ganglioneuroma, islet cell tumor, Kaposi'ssarcoma, kidney cancer, larynx cancer, leiomyoma tumor, liver cancer,malignant carcinoid, malignant hypercalcemia, malignant melanomas,marfanoid habitus tumor, medullary carcinoma, metastatic skin carcinoma,mucosal neuromas, mycosis fungoide, neck cancer, neural tissue cancer,neuroblastoma, osteogenic sarcoma, osteosarcoma, ovarian tumor, pancreascancer, parathyroid cancer, pheochromocytoma, primary brain tumor,rectum cancer, renal cell tumor, retinoblastoma, rhabdomyosarcoma,seminoma, skin cancer, small-cell lung tumor, non-small cell lung cancer(NSCLC), soft tissue sarcoma, squamous cell carcinoma, stomach cancer,thyroid cancer, topical skin lesion, veticulum cell sarcoma, Wilm'stumor, and/or combinations thereof.

In one embodiment, the graphene oxide (28) is functionalized withpolyethylene glycol. For example, expandable graphite may be exfoliatedand heated at about 900° C. for about 1 hour under argon to removeintercalated acid molecules. Then, a salt such as NaCl may be added andremoved by filtration with water to reduce particle size. Then, a strongacid, such as sulfuric acid, may be added to effect intercalation.Further, an oxidizing agent such as KMnO₄ may be added and the productmay be washed. Subsequently, carboxylic acid functional groups made beadded along with NaOH followed by sonication, neutralization, filteringand washing. The product formed is then typically a carboxylic acidmodified graphite oxide (GO—COOH). This product may be then sonicatedwith a 6-arm polyethylene glycol-amine andN-(3-dimethylaminopropyl-N′-ethylcarbodiimide) hydrochloride may beadded. Finally, mercaptoethanol may be added and the product subjectedto centrifugation in PBS to form NGO-PEG.

In still other embodiments, the graphene oxide (28) is functionalizedwith a linking molecule (36), e.g. a linker such as GMBS which is knownas N-[γ-maleimidobutyryloxy]succinimide ester in the art. The linkingmolecule (36) is not particularly limited. It is also contemplated thatthe graphene oxide (28) and/or the linking molecule (36) may befunctionalized or bonded to a marker (38) such as a protein such asNeutrAvidin, see e.g. FIGS. 12A/B and 14A/B. The protein may be directlybonded to the graphene oxide (28) and/or the linking molecule (36). Itis further contemplated that the graphene oxide (28), the linkingmolecule (36), and/or the marker (38) may be functionalized or bonded toan antibody (40) such as EpCAM against the EpCAM antigen expressed onthe surface of cancer cells. The antibody (40) may be directly bonded tothe graphene oxide (28), the linking molecule (36), and/or the marker(38). In one embodiment, as shown in FIGS. 1A and 1B, the graphene oxide(28) is disposed on a plurality of extensions (26), is functionalizedwith (i.e., bonded to) a linking molecule (36) which, in turn, isfunctionalized with (i.e., bonded to) a protein which, also in turn, isfunctionalized with (i.e., bonded to) an antibody (40). The antibody(40) can then bind a rare cell (22) such as a CTC. The instantdisclosure is not limited to the aforementioned antibodies, proteins,etc. and one or more known in the art may be utilized and bonded to thegraphene oxide (28). Suitable non-limiting examples include variousantibodies and/or proteins, epithelial surface markers such as EGFR,prostate markers such as PSMA, PSA, cancer cell markers such as CD133,CD44, ALDH, endothelial markers such as CD31, CD34, leukocyte markerssuch as CD45, CD4, exosome/microvessicle markers such as CD63, etc.Alternatively, various peptides recognizing particular DNA sequences maybe utilized.

In one embodiment, the graphene oxide is functionalized with a bindingagent, the binding agent includes the reaction product ofphospholipid-polyethylene-glyco-amine (PL-PEG-NH₂) andN-γ-maleimidobutyryloxy succinimide ester (GMBS), the reaction productis further bonded to a protein, and the protein is further bonded to anantibody for interaction with the rare cells.

Additional Non-Limiting Embodiments of a Microfluidic Device:

In one embodiment, the system (20) is further defined as a microfluidicdevice. The microfluidic device (and/or system (20)) may include amicrofluidic channel (32) and/or a microfluidic chamber (56) throughwhich blood, body fluids, and/or other substances can flow. Twonon-limiting examples of suitable microfluidic channels (32) are setforth in FIGS. 2D and 2E. Typically, larger devices include microfluidicchambers (56) as opposed to microfluidic channels (32), but this is notnecessarily true in every embodiment. The microfluidic device (and/orsystem (20) and/or microfluidic channel (32) and/or microfluidic chamber(56)) may also include one, two, or a plurality of posts (54), such asPDMS posts, to support one or more chambers, channels, or layers, seee.g. FIG. 3.

In one embodiment, the microfluidic device has one or more microfluidicchannels (32) and/or chambers (56) one or more of which eachindependently having a length, height, and/or width of from 1 μm to 1000μm (i.e., 1 mm) In various embodiments, one or more of these values isfrom 1 μm to 100, 5 to 95, 10 to 90, 15 to 85, 20 to 80, 25 to 75, 30 to70, 35 to 65, 40 to 60, 45 to 55, or 50 to 55 μm. In other embodiments,one or more of these values is from 100 to 1000, from 125 to 975, from150 to 950, from 175 to 925, from 200 to 900, from 225 to 875, from 250to 850, from 275 to 825, from 300 to 800, from 325 to 775, from 350 to750, from 375 to 725, from 400 to 700, from 425 to 675, from 450 to 650,from 475 to 625, from 500 to 600, from 525 to 575, or from 550 to 575μm. In other embodiments, the width may be up to 5 mm, and the length upto 100 to 1000 mm. The dimensions of the microfluidic device, as awhole, are not particularly limited. In various embodiments, e.g. as setforth in the Figures, L₂ and L₃ may be from 5 to 100 mm, W₂ and W₃ maybe from 5 to 50 mm, and T₁ and T₂ may be from 100 μm to 10 mm Any of theaforementioned values may, for example, vary by 1, 2, 3, 4, 5, 10, 15,20, or 25+% in varying non-limiting embodiments. All values, and rangesof values, between and including the aforementioned values are alsohereby expressly contemplated in various non-limiting embodiments.

One of more microfluidic channels (32) and/or chambers (56) may eachindividually have a unique shape and/or structure. In addition, onemicrofluidic channel (32) and/or chamber (56) may have a shape orpattern different from another microfluidic channel (32) and/or chamber(56) in the same device. The geometry of these patterns is also notparticularly limited. The patterns may be geometric, non-geometric,uniform or non-uniform, e.g. straight, zig-zag, herringbone, circular oroval, triangular, whorl-shaped, ribbon-shaped, marble, spiral-shaped,coil-shaped, curl-shaped, twisted, looped, helix, serpentine,sinusoidal, winding, and/or random, and the like.

Suitable but non-limiting microfluidic devices are described inWO2009/051734 and PCT/US10/53221, each of which is expresslyincorporated herein by reference in non-limiting embodiments. Additionalsuitable but non-limiting microfluidic devices are set forth in FIGS.2A-2G. Other suitable, but non-limiting, microfluidic devices aredescribed in S. Wang et al., “Highly Efficient Capture of CirculatingTumor Cells by Using Nanostructured Silicon Substrates with IntegratedChaotic Micromixers,” Angewandte Chemie, vol 50, pp. 3084-3088, 2011,which is expressly incorporated herein by reference in non-limitingembodiments. As described in this reference, it is contemplated thatthis disclosure may utilize silicon nanopillars and/or PDMS channels.

The microfluidic device may include one or more walls (30) orientedsubstantially perpendicularly, or transversely, to a floor (42),supplemental layers (34), microfluidic channels (32) and/or microfluidicchambers (56). As set forth in FIG. 1A, the microfluidic device may alsohave a central body (44), a longitudinal axis (L₁), and upstream anddownstream ends (46, 48) opposite each other, wherein the central body(44) defines the microfluidic channel (32) and/or microfluidic chambers(56) which is in fluid communication with the upstream and downstreamends (46, 48) along the longitudinal axis (L₁) for receiving the sample.The microfluidic device may also include an entrance (50) (i.e. inlet)defined by the central body (44) and disposed at the upstream end (46)of the central body and include an exit (52) (i.e., outlet) also definedby the central body (44) and disposed at the downstream end (48) of thecentral body (44) wherein both the entrance (50) and exit (52) aredisposed transverse to the longitudinal axis (L₁).

The geometry of the microfluidic channel (32) and the one or more walls(30) is not particularly limited but may be designed to increase ordecrease flow through, velocity through, or pressure in, themicrofluidic channel (32).

The microfluidic device may include a single microfluidic channel (32)and/or chamber (56), two microfluidic channels (32) and/or chambers(56), or three or more (i.e., a plurality of) microfluidic channels (32)and/or chambers (56). The microfluidic channels (32) and/or chambers(56) can be arranged in series, in parallel, or in any geometric orpuzzle configuration as selected by one of skill in the art. In oneembodiment, one or more microfluidic channels (32) and/or chambers (56)are arranged in an approximate herringbone pattern. Each individualmicrofluidic channel (32) and/or chamber (56) may be used to isolate oneor more types of material or rare cells (22). In various embodiments, asample of blood, bodily fluid, etc. is segmented into two or moresegments and the segments flow through different microfluidic channels(32) and/or chambers (56) at one or more pressures and/or velocities.

The microfluidic device may be designed to allow for optical or visualinspection of the microfluidic channels (32) and/or microfluidicchambers (56). For example, the microfluidic device may include a top(58), bottom (62), and/or side (60), e.g. as set forth in FIG. 2C, whichmay be transparent to allow for optical or visual inspection.Alternatively, the microfluidic device may include a top, bottom, and/orside which may be opaque. It is also contemplated that the microfluidicdevice may not include a top.

In addition, the microfluidic device may be designed to maximizeefficiency relative to flow, velocity and/or shear force of a samplepassing therethrough. In various embodiments, the maximum shear forceexerted on a cell, based on a volumetric flow rate of about 1 mL/h, isabout 0.4 dynes/cm² at θ=68°, and the maximum velocity is about 460μm/s. The shear stress produced in a microfluidic channel (32) and/ormicrofluidic chamber (56) is typically of from about 0.1 to about 20dyn/cm² and may be less than 15, 10, 5, 1, or 0.5, dyn/cm². Shear stressis not necessarily constant throughout a microfluidic channel (32). Inother embodiments, a sample may be transported through the microfluidicchannel (32) and/or chamber (56) at a rate of 0.1 mL to 30 mL/hr.Typical flow rates are typically from 0.5 to 1, from 1 to 20, 2 to 19, 3to 18, 4 to 17, 5 to 16, 6 to 15, 7 to 14, 8 to 13, 9 to 12, or 10 to11, mL/hr. However, these rates are not limiting and the rate at whichthe sample passes through may be greater or less than those describedabove. Any of the aforementioned values may, for example, vary by 1, 2,3, 4, 5, 10, 15, 20, or 25+% in varying non-limiting embodiments. Allvalues, and ranges of values, between and including the aforementionedvalues are also hereby expressly contemplated in various non-limitingembodiments.

The volume of the microfluidic channel (32) and/or microfluidic chamber(56) may be customized depending on a volume of the sample used. Thevolume of the microfluidic channel (32) and/or microfluidic chamber (56)may be smaller or larger than the size of the sample or may beapproximately the same as the size of the sample. In variousembodiments, the microfluidic device and/or the microfluidic channel(32) and/or microfluidic chamber (56) has a volume of from about 10 μLto 20 mL, from about 100 μL to 15 mL, from about 100 μL to 10 mL, fromabout 100 μL to 5 mL, from about 100 μL to 1 mL, or from about 100 μL to0.5 mL. However, these volumes are not limiting and the volume of themicrofluidic device and/or the microfluidic channel (32) and/ormicrofluidic chamber (56) may be greater or less than those describedabove. Any of the aforementioned values may, for example, vary by 1, 2,3, 4, 5, 10, 15, 20, or 25+% in varying non-limiting embodiments. Allvalues, and ranges of values, between and including the aforementionedvalues are also hereby expressly contemplated in various non-limitingembodiments.

The microfluidic channel (32) and/or microfluidic chamber (56) may bemodified to increase surface area, volume, etc. to increase aprobability that a rare cell with be captured. For example, when thewalls (30) are substantially planar, the height of the microfluidicchannel (32) and/or microfluidic chamber (56) may be designed so thatrare cells are more efficiently detected and/or trapped.

The microfluidic device is not particularly limited to any particularefficiency. However, in various embodiments, the microfluidic device cantypically identify, enumerate, detect, capture, and/or isolate from 1 to10,000, 1 to 7,500, 1 to 5,000, 1 to 2,500, 1 to 1500, from 5 to 1000,from 10 to 500, from 25 to 200, or from 50 to 100, rare cells (22) froma blood sample of about 1 mL or less. Alternatively, the system (20)and/or microfluidic device may have a rare cell capture efficiency of atleast 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 99, percent determined as (rare cells captured on the(functionalized) graphene oxide (or any protein, antibody, marker, etc.bound thereto) divided by a total number of rare cells introduced to thesystem and/or microfluidic device) multiplied by 100. In otherembodiments, the system (20) and/or microfluidic device may have a rarecell capture efficiency of 95 to 100, 90 to 95, 90 to 100, 85 to 95, 85to 90, 80 to 85, 80 to 90, 80 to 95, 75 to 80, 75 to 85, 75 to 90, 75,to 95, 70 to 75, 70 to 80, 70 to 85, 70 to 90, 75 to 95, 50 to 95, 55 to90, 60 to 85, 65 to 80, 65 to 75, 65 to 70, 25 to 50, 59 to 75, or 25 to75, percent, as determined using the formula described immediatelyabove. In various embodiments, the microfluidic device has a rare cellcapture efficiency of about 70, 75, or 80 plus or minus about 20, 25, or30, at 5-20 cells/mL spiked in blood. Any of the aforementioned valuesmay, for example, vary by 1, 2, 3, 4, 5, 10, 15, 20, or 25+% in varyingnon-limiting embodiments. All values, and ranges of values, between andincluding the aforementioned values are also hereby expresslycontemplated in various non-limiting embodiments.

In various embodiments, the microfluidic device and system may capture,on average, about 155±236 CTCs/mL for NSCLC, about 16 to 292 CTCs/mL formetastatic prostate, about 25 to 174 CTCs/mL for localized prostatecancer, about 9 to 831 CTCs/mL for pancreatic cancer cells, about 5 to176 CTCs/mL for breast cancer cells, and about 42 to 375 (121±127)CTCs/mL for colorectal cancer cells. The microfluidic device may allowcaptured cells to be grown and cultivated, see e.g. FIGS. 17 and 22-24,and/or washed such that non-specifically bound cells, e.g., leukocytes,may be removed which may result in about a 10⁶-fold enrichment. Any ofthe aforementioned values may, for example, vary by 1, 2, 3, 4, 5, 10,15, 20, or 25+% in varying non-limiting embodiments. All values, andranges of values, between and including the aforementioned values arealso hereby expressly contemplated in various non-limiting embodiments.

The microfluidic device may also include or be coupled to one or morecomponents such as reservoirs, pumps, valves, actuators, pipes, tubes,electrodes, meters, computers, electronic monitoring devices, analyticaldevices, electrical potential and/or resistance monitoring devices, andthe like. Those of skill in the art may select one or more of thecomponents to couple to the microfluidic device.

In one embodiment, the microfluidic device includes a silicon substratethat includes approximately has 60,000 extensions (e.g. formed fromgold) disposed in leaf patterns having a size of 100 μm×100 μm. Thedistance between each extension in a column, in this embodiment, is 150μm. The overall size of the microfluidic device of this embodiment isabout 24.5 mm×60 mm×3 mm. The microfluidic device of this embodimentalso includes a supplemental layer (34) that is PDMS and that has amicrofluidic chamber therein wherein the chamber has a height of 50 μmand a volume of 45 μL. Graphene oxide nanosheets may be self-assembledonto the extensions, in this embodiment, at a thickness of about 100 nm.The graphene oxide of this embodiment may also be functionalized withEpCAM antibodies. It is contemplated that the aforementioneddescriptions and characteristics are not necessarily limited to thisparticular embodiment and may apply to other embodiments describedherein.

The System (120)

Substrate:

The substrate (124) is not particularly limited and may be furtherdefined as being, including, consisting essentially of, or consistingof, a metal, plastic, polymer, inorganic compound, glass, silicon (e.g.—Si—Si—), silicone (e.g. —Si—O—Si— or PDMS), epoxy, semiconductors,and/or combinations thereof. The terminology “consist essentially of”typically describes that the substrate (124) includes one or more of theparticular aforementioned materials and is free of, or includes lessthan 0.1 or 1, weight percent, of dissimilar materials. The substrate(124) may be fabricated using any technique known in the art including,but not limited to, molding, photolithography, electroforming,machining, chemical vapor deposition, and the like.

The substrate (124) may also be further defined as a device, layer,film, coating, sheet, skin, chip, block, or wafer. In variousembodiments, the substrate (124) is further defined as a tri-layeredsubstrate that includes a silicon layer, an SiO₂ layer, and a PDMS(i.e., polydimethylsiloxane) layer. Alternatively, the substrate (124)may be further defined as a single layer. In one embodiment, additionallayers, e.g. the SiO₂ layer and the PDMS layer, are disposed on thesingle layer and may be individually described as one or moresupplemental (or support) layers. Depending on overall design and shape,one or more of the substrate (124) and/or one or more supplementallayers may be independently further defined as an outermost layer, aninnermost layer, or an interior layer, e.g. of a device or of the system(120). In other embodiments, the substrate (124) and/or one or moresupplemental layer may be, include, consist essentially of, or consistof, one or more of polyethylene terephthalate (PET), polyimide,polyether ether ketone (PEEK), and/or combinations thereof.

The substrate (124) and/or the one or more supplemental layers may bebonded together by any means known in the art including use ofadhesives, chemical bonding techniques, and physical bonding techniques.In one embodiment, the substrate (124) includes an SiO₂ layer that isbonded to the substrate (124) using oxygen plasma treatment.

The substrate (124) and one or more supplemental layers are not limitedto any particular configuration or structure and each of the substrate(124) and one or more supplemental layers may independently be disposedin any order or configuration relative to one another. All combinationsof these layers and configurations are herein expressly contemplated.Each of the substrate (124) and supplemental layers are also notparticularly limited to any particular cross-section and each mayindependently have, but is not limited to having, a rectangularcross-section, a square cross-section, a triangular cross-section, acircular or oval cross-section, an “I”-shaped cross-section, a“C”-shaped cross-section, an “L”-shaped cross-section, a “T”-shapedcross-section, a “U”-shaped cross-section, a “W” shaped cross-section,or a hexagonal cross section. As shown in FIG. 26, the substrate (124)has a hexagonal cross section. The substrate (124) (and supplementallayers if present) may be solid, hollow, or have solid sections andhollow sections. Further, and as shown at least in FIGS. 26 and 27, thesubstrate (124) may be placed on a support (125), such as a glass slide.The support (125) may have any suitable shape (such as a rectangularshape, a circular shape, an oval shape, a square shape, etc.) and mayhave any suitable dimensions. In an embodiment, the support (125) is a 2inch by 3 inch glass slide.

As shown in FIGS. 26-28, the substrate (124) has an outer edge (127) anddefines a center axis A. The center axis A may be defined at the centerof the substrate (124) regardless of the shape of substrate (124). In anembodiment, the center axis A may be positioned at the center of thesubstrate (124). In another embodiment, the center axis A may be offsetfrom the center of the substrate (124). The outer edge (127) may bedefined along the outer periphery of the substrate (124). In anembodiment, the outer edge (127) may include a single outer edge, suchas the outer edge of a circular-shaped or oval-shaped substrate (124).In another embodiment, the outer edge (127) may include a plurality ofouter edges, such as three outer edges for a triangular-shaped substrate(124), four outer edges for a rectangular-shaped substrate (124), fiveouter edges for a pentagonal-shaped substrate (124), etc.

In an embodiment, and as shown in FIGS. 26-28, the system (120) furtherincludes a fluid inlet defined in the substrate (124) for receiving afluid, such as a bodily fluid, e.g., blood. The fluid inlet may bedefined at the center of the substrate (124), such as along the centeraxis A. The fluid inlet may alternatively be offset from the center ofthe substrate (124) and in turn may be offset from the center axis A.The system (120) further includes at least one fluid outlet defined inthe substrate (120) for allowing the fluid to exit the system (120). Inthe embodiment shown in FIGS. 26-28, the fluid outlet may be defined atone or more positions along the outer edge (127) of the substrate (124).As described in further detail below, and with reference to FIG. 30, thefluid enters the system (120) through the inlet at or near the center ofthe substrate (124), flows radially toward the outer edge (127), andexits the system (120) through the fluid outlet(s).

The overall size of each of the substrate (124) (and supplementallayers) is not particularly limited. In one embodiment, the substrate(124) has dimensions of about 35 mm×10 mm×3 mm. However, thesedimensions are not limiting and may vary. Suitable non-limiting examplesof substrates (124) and supplemental layers (134) have length, width,and height dimensions on the scale of 1 to 100, 1 to 75, 1 to 50, 1 to25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 0.1 to 1, inches, centimeters,and/or millimeters. Any of the aforementioned values may, for example,vary by 1, 2, 3, 4, 5, 10, 15, 20, or 25+% in varying non-limitingembodiments. All values, and ranges of values, between and including theaforementioned values are also hereby expressly contemplated in variousnon-limiting embodiments. It is also contemplated that a microfluidicdevice, as described in greater detail below, may have the same ordifferent dimensions from one or more of the substrate (124) and/or thesupplemental layer(s).

Extension:

The system (120) also includes the extension (126) coupled to thesubstrate (124), and extending outwardly from the substrate (124). Theterminology “extension” may describe a single extension, two extensions,or a plurality of extensions, in various embodiments, throughout. Saiddifferently, whenever the terminology “extension” is used, thatterminology may describe various embodiments including a singleextension, two extensions, or a plurality of extensions. In the presentembodiment, and as shown in FIGS. 26 and 29, the system (120) comprisesa plurality of extensions (126) extending outwardly from said substrate(124) and radially arranged about said center axis (A) of the substrate(124).

As shown, e.g., in FIG. 26, the extensions (126) may extend outwardlyfrom the substrate (124) approximately perpendicularly to an axis (L₁)or may extend outwardly at another angle to the substrate (124) and/orthe axis (L₁), e.g. at an obtuse or acute angle, such as 30°, 45°, or60°. The extensions (126) may be coupled to the substrate (124) via anymeans known in the art such as through chemical and physicalconnections, e.g. with adhesives, via chemical bonding, and the like.Similarly, the extensions (126) may be coupled to the substrate (124) indirect contact with the substrate (124) or in indirect contact with thesubstrate (124), e.g. separated by one or more layers, compounds,molecules, etc. As an additional example, the extensions (126) may bedisposed in direct contact with an intermediate or supplemental layer orconnection which, in turn, may be disposed either directly or indirectlywith the substrate (124). It is contemplated that the extension (126)may still be coupled to the substrate (124) even though there is nodirect contact therebetween.

The extensions (126) are arranged about the center axis (A) of thesubstrate (124). In an embodiment, and as shown in FIG. 26, theextensions (126) may be radially arranged about the center axis (A) ofthe substrate (124). Further, the system (120) may include the pluralityof extensions (126) arranged about the center axis (A) in at least onerow (R). As shown in FIG. 26, for example, the system (120) may includethe plurality of extensions (126) arranged radially about the centeraxis (A) in multiple rows (R). For instance, a first row (R₁) ofextensions (126) may be radially arranged about the center axis (A), asecond row (R₂) of extensions (126) may be radially arranged about thecenter axis (A) behind the first row of extensions (126), a third row(R₃) of extensions (126) may be radially arranged about the center axis(A) behind the second row of extensions (126), and a fourth row (R₄) ofextensions (126) may be radially arranged about the center axis (A)behind the third row (R₃). While FIG. 26 shows four rows (R₁, R₂, R₃,R₄), it is to be appreciated that the system (120) may include anynumber of rows (R), such as two, three, four, five, six, seven, etc.rows (R) of extensions (126).

With reference to FIGS. 26 and 29, the extensions (126) may be radiallyarranged about the center axis (A) of the substrate (124) in a pluralityof rows (R) to define the channel (132) enabling the fluid to moveradially from the center axis (A) toward the outer edge (127) of thesubstrate (124). In an embodiment, the channel (132) is defined betweenadjacent extensions (126) of each of the plurality of rows (R). Forinstance, a channel (132) may be defined between adjacent extensions(126) in a single row (such as, e.g., the first row (R₁), the second row(R₂), etc.) of extensions (126). A channel (132) may also be definedbetween extensions (126) of adjacent rows (R) (such as, e.g., a firstextension (126) in the first row (R₁) and a second extension (126) inthe second row (R₂) which is adjacent to the first extension (126)). Thechannels (132) defined between adjacent extensions (126) of each of theplurality of rows (R) and the channels (132) defined between extensions(126) of adjacent rows (R) may be interconnected to form a singlechannel (132) through which fluid flows from the inlet toward theoutlet(s) of the system (120). In an example, and as described infurther detail below, fluid (such as bodily fluid) flows through theinterconnected channels (132) and rare cells (such as cancer cells) inthe bodily fluid contact and interact with the extensions (126) radiallyarranged about the center axis (A).

In an embodiment, each of the extensions (126) is spaced from anadjacent one of the extensions (126) (in the same row (R) or in adjacentrows (R)) a distance of from 10 to 100 μm. In another example, each ofthe extensions (126) is spaced from an adjacent one of the extensions(126) a distance of from 20 to 50 μm. In still another example, each ofthe extensions (126) is spaced from an adjacent one of the extensions(126) a distance of from 26 to 32 μm. Any of the aforementioned valuesmay, for example, vary by 1, 2, 3, 4, 5, 10, 15, 20, or 25+% in varyingnon-limiting embodiments. All values, and ranges of values, between andincluding the aforementioned values are also hereby expresslycontemplated in various non-limiting embodiments.

The extensions (126) may be disposed on any one or more portions orsegments of the substrate (124) or microfluidic device. In the presentembodiment, the plurality of extensions (126) is disposed on or in thesubstrate (124) or microfluidic device in a radial pattern describedabove. Further, a total number of extensions (126) may vary and, in someembodiments, may exceed hundreds, thousands, hundreds of thousands,millions, tens of millions, etc. It is to be appreciated that the totalnumber of extensions (126) is not particularly limited.

Each extension (126) typically has an upper end (154) and a lower end(155) and a vertical axis (V) that extends through the upper and lowerends (154, 155), as shown in FIG. 32. Typically, the upper and lowerends (154, 155) extend along the vertical axis (V). The extension (126)also typically has a horizontal axis (H₁) that extends between the upperand lower ends (154, 155), as also shown in FIG. 32. At least one of theextensions (126) may be bean-shaped having a concave side (133) with anarc (135). In an embodiment, each of the extensions (126) extendingoutwardly from the substrate (124) are bean-shaped. Examples of thebean-shaped extensions (126) are shown at least in FIGS. 26, 29, and31A/B. In an example, the arc (135) has an arc angle of from 75 to 90degrees. In another example, the arc (135) has an arc angle of from 80to 90 degrees. In yet another example, the arc (135) has an arc angle ofabout 95 to 90 degrees. In another example, the arc (135) has an arcangle of about 90 degrees. Any of the aforementioned values may, forexample, vary by 1, 2, 3, 4, 5, 10, 15, 20, or 25+% in varyingnon-limiting embodiments. All values, and ranges of values, between andincluding the aforementioned values are also hereby expresslycontemplated in various non-limiting embodiments.

The extensions (126) may be disposed substantially perpendicularly tothe substrate (124) and/or horizontal axis (H₁) or disposed transversely(i.e., at any angle) to the substrate (124) and/or horizontal axis (H₁).It is also contemplated that the extensions (126) may be disposed suchthat the horizontal axis (H₁) is disposed approximately parallel to, ortransverse to, the substrate (124). Each of the extensions (126) may befurther defined as a post or rod, e.g. a micro-post, micro-rod,nanopost, nanorod, etc. In one embodiment, each of the extensions (126)is further defined as an electrode. Typically, the extension (126) hasmicro- or nano-scale dimensions.

In various embodiments, each of the extensions (126), e.g. a nanopost,has a height (e.g. H₂) of about 1 to 5 nm and a width (e.g., W₂) ofabout 40 to 60 μm. In other embodiments, each extension (126) has aheight (H₂) of about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 nm, or rangesthereof. In other embodiments, each extension (126) has a width (e.g.W₂) of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, or μm, or ranges thereof. In an example, the width (W₂)of each extension (126) may include a first width (W_(2A)) extendingfrom the center of the arc to a first end (131) of the extension (126)and a second width (W_(2B)) extending from the center of the arc to asecond end (133) of the extension (126). In an example, the first width(W_(2A)) of the extension (126) ranges from 100 to 250 μm, from 150 to200 μm, or from 175 to 195 μm, or ranges thereof, and the second width(W_(2B)) of the extension (126) ranges from 100 to 250 μm, from 150 to200 μm, or from 175 to 195 μm, or ranges thereof. In an example, thefirst (W_(2A)) and second (W_(2B)) widths are substantially equal. Inanother example, the first (W_(2A)) and second (W_(2B)) widths aredifferent. Any of the aforementioned values may, for example, vary by 1,2, 3, 4, 5, 10, 15, 20, or 25+% in varying non-limiting embodiments. Allvalues, and ranges of values, between and including the aforementionedvalues are also hereby expressly contemplated in various non-limitingembodiments.

The extensions (126) may be, include, consist essentially of, or consistof, a plastic, polymer (such as polymethylmethacrylate (PMMA)) or metalor combinations thereof. In one embodiment, the metal is gold (e.g. theextension (126) may be formed from gold). Alternatively, the metal maybe, include, consist essentially of, consist of, or be chosen from thegroup of, transition metals, precious metals, rare earth metals, andcombinations thereof. In various embodiments, it is contemplated thatthe extension (126) be, include, consist of, or consist essentially of,a metal, such as gold, silver, and/or copper, and/or a mixed metalcompound such as indium-tin oxide (ITO). The terminology “consistessentially of” typically describes that the extension (126) includesone or more of the aforementioned materials and is free of, or includesless than 0.1 or 1, weight percent, of a non-metal or a non-mixed metalcompound or another of the aforementioned materials.

The extensions (126) may be formed by any method known in the art. Inone embodiment, the extensions (126) are formed by evaporating andpatterning metal layers, e.g. Cr/Au layers (10/100 nm). In variousembodiments, the extensions (126) can be formed using a lift-off processwhich typically allows for fine patterns to be formed. A photoresist maybe coated on a silicon substrate (124) and patterned byphotolithography, see e.g. FIG. 5. Then metal layers may be deposited onthe silicon wafer. Subsequently, the substrate (124) may be immersed inacetone or a photoresist remover solution. A patterned gold layertypically remains. In other embodiments, a shadow mask can be used inconjunction with depositing a layer, e.g. a gold layer. Electroplatingtechniques may also be utilized throughout this disclosure.

Further details of the extensions (126) and their arrangement on thesubstrate (126) and forming the system (120) or microfluidic device aredescribed below.

Functionalized Graphene Oxide:

As shown in FIG. 33, the functionalized graphene oxide (128) may bedisposed on each of the extensions (126). The graphene oxide (128),either pre- or post-functionalization, may be disposed on, or attachedto, the extensions (126) by any means known in the art including bothphysical and chemical attachment including covalent bonding,electrostatic attraction, etc. In one embodiment, the extensions (126)are exposed to a compound, such as TBA Hydroxide, that facilitatesbinding of the graphene oxide (128) to the extensions (126). Withoutintending to be bound by any particular theory, it is believed that TBAHydroxide forms a cation (+) that interacts with anions (−) (e.g. goldanions) of the extensions (126).

The extensions (126) may include one or more types of (functionalized)graphene oxide (128) and one or more markers, binding agents, etc.,bonded or attached thereto, see e.g. FIGS. 16A/B. For example, multiplebinding agents may bind to the same or different cells and may be placedin the same or different microfluidic channels (132) or on the same ordifferent extensions (126).

Graphene oxide is a single layer form of graphite oxide and can befurther defined as a form of graphene that includes oxygen functionalgroups on basal planes and edges. Typically, graphene oxide is describedas a strong paper-like material. Graphene oxide, whether functionalizedor not functionalized, may have a thickness of about 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0nm, or up to 50 nm, e.g. in tenth- or half-nanometer increments, each±0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0, nm, see e.g. FIGS.9A-D. In various embodiments, the combined height of the(functionalized) graphene oxide and the extension is from 1 μm to 1000μm (i.e., 1 mm) In various embodiments, the combined height is from 1 μmto 100, 5 to 95, 10 to 90, 15 to 85, 20 to 80, 25 to 75, 30 to 70, 35 to65, 40 to 60, 45 to 55, or 50 to 55, μm. In other embodiments, thecombined height is from 100 to 1000, from 125 to 975, from 150 to 950,from 175 to 925, from 200 to 900, from 225 to 875, from 250 to 850, from275 to 825, from 300 to 800, from 325 to 775, from 350 to 750, from 375to 725, from 400 to 700, from 425 to 675, from 450 to 650, from 475 to625, from 500 to 600, from 525 to 575, or from 550 to 575 μm. Any of theaforementioned values may, for example, vary by 1, 2, 3, 4, 5, 10, 15,20, or 25+% in varying non-limiting embodiments. All values, and rangesof values, between and including the aforementioned values are alsohereby expressly contemplated in various non-limiting embodiments.

In one embodiment, the graphene oxide is formed from graphite, asdescribed in D. Li, M. B. Muller, S. Gilje, R. B. Kaner, and G. G.Wallace, “Processable aqueous dispersions of graphene nanosheets,”Nature Nanotechnology, vol. 3, pp. 101-105, 2008, which is expresslyincorporated herein by reference in a non-limiting embodiment. Inanother embodiment, the graphene oxide is formed using the procedure asdescribed in Z. Wei, D. E. Barlow, and P. E. Sheehan, “The Assembly ofSingle-Layer Graphene Oxide and Graphene Using Molecular Templates,”Nano Letters, Vol. 8, No. 10, pp. 3141-3145, 2008, also expresslyincorporated herein by reference in a non-limiting embodiment. In stillanother embodiment, the graphene oxide is formed from graphene sheetsthat are formed using the procedure as described in H. Wang, X. Wang, X.Li, H. Dai, “Chemical Self-Assembly of Graphene Sheets,” Nano Research,Vol. 2, pp. 336-342, 2009, also expressly incorporated herein byreference in a non-limiting embodiment. In even another embodiment, thegraphene oxide is formed using the procedure described in X. Sun, Z.Liu, K. Welsher, J. T. Robinson, A. Goodwin, S. Zaric, H. Dai“Nano-Graphene Oxide for Cellular Imagine and Drug Delivery” NanoResearch, Vol. 1, pp. 203-212, 2008, also expressly incorporated hereinby reference in a non-limiting embodiment. It is also contemplated thatthe graphene oxide may be formed using the procedure described in U.S.Pat. App. Pub. No. US 2010/0028681, which is also expressly incorporatedherein by reference in a non-limiting embodiment.

In one embodiment, graphene oxide sheets are formed byexfoliation-reintercalation-expansion methods, as described above. Inanother embodiment, ground natural graphite is intercalated by oleum inthe presence of sodium nitrate. The product may then be treated with anaqueous solution of tetrabutylammonium (TBA) hydroxide and suspended byPL-PEG-NH₂ in DMF.

In still other embodiments, the graphene oxide sheets can be formed onthe surface of the extensions (126). Without intending to be bound byany particular theory, it is believed that selective absorption ofgraphene sheets onto the extensions (126), e.g. gold patterns, can occurvia electrostatic interactions. Suitable, but non-limiting, grapheneoxide (128) sheets can have sizes, e.g. length and/or width, from about10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425,450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775,800, 825, 850, 875, 900, 925, 950, or 975 nm, to about 1 micrometer.Alternatively, the upper range of the length and/or width may be up to100 micrometers, e.g. in increments of, e.g. half, tenth, or hundredths,or thousandths, of a micrometer. Any of the aforementioned values may,for example, vary by 1, 2, 3, 4, 5, 10, 15, 20, or 25+% in varyingnon-limiting embodiments. All values, and ranges of values, between andincluding the aforementioned values are also hereby expresslycontemplated in various non-limiting embodiments.

The graphene oxide (128) that is functionalized typically forms stablesuspensions in water and can aggregate in salt or other biologicalsolutions. In various embodiments, the graphene oxide (128) isfunctionalized with one or more functional groups including, but notlimited to, aliphatic groups, aromatic groups, nitrogen including groupssuch as amines and amides, carboxyl groups, sulfur including groups,phosphorous including groups, and the like. Alternatively, the grapheneoxide (128) can be functionalized with one or more markers, antibodies,antigens, proteins, tumor specific binding agents (e.g. anti-EpCAM), andthe like. In another embodiment, the terminology “tumor specific bindingagent” describes an agent that binds to a nonhemopoietic cell that canform a tumor, such as a cell not of hemopoietic origin, excluding bloodcells and immune cells, but including epithelial cells, endothelialcells, neurons, hepatocytes, nephrons, glial cells, muscle cells, skincells, adipocytes, fibroblasts, chondrocytes, osteocytes, andosteoblasts. The binding agent may bind to a cell surface marker that isspecific for a type of cell that can form a tumor and that is notnormally found in circulating blood. In an alternative, the bindingagent may bind to a cell surface marker that is specific for atransformed cell. Such agents may also bind to healthy cells circulatingin blood from non-pathogenic origins, e.g., venipuncture or trauma. Inother embodiments, Streptavidin and/or one or more antibodies forvarious viruses may be utilized.

In various embodiments, the graphene oxide (128) is functionalized withone or more markers that allows for identification, enumeration,detection, capture, and/or isolation of genomic DNA, cDNA, or mRNAsequences, proteins or other intracellular contents that are indicativeof a type or presence of a particular tumor, determination of thepresence or absence of certain mutations in EGFR, HER2, prostatespecific antigen TMPRSS2-ERG, CD133, CD44, CD24, epithelial-specificantigen (ESA), Nanog, 25 BMI1, and the like. Alternatively, the grapheneoxide (28) may be functionalized with one markers that allows foridentification, enumeration, detection, capture, and/or isolation ofcells related, but not limited, to one or more of the following cancers:ostate, lung, adenocarcinoma, adenoma, adrenal cancer, basal cellcarcinoma, bone cancer, brain cancer, breast cancer, bronchi cancer,cervical dysplasia, colon cancer, epidermoid carcinoma, Ewing's sarcoma,gallbladder cancer, gallstone tumor, giant cell tumor, glioblastomamultiforma, head cancer, hyperplasia, hyperplastic corneal nerve tumor,in situ carcinoma, intestinal ganglioneuroma, islet cell tumor, Kaposi'ssarcoma, kidney cancer, larynx cancer, leiomyoma tumor, liver cancer,malignant carcinoid, malignant hypercalcemia, malignant melanomas,marfanoid habitus tumor, medullary carcinoma, metastatic skin carcinoma,mucosal neuromas, mycosis fungoide, neck cancer, neural tissue cancer,neuroblastoma, osteogenic sarcoma, osteosarcoma, ovarian tumor, pancreascancer, parathyroid cancer, pheochromocytoma, primary brain tumor,rectum cancer, renal cell tumor, retinoblastoma, rhabdomyosarcoma,seminoma, skin cancer, small-cell lung tumor, non-small cell lung cancer(NSCLC), soft tissue sarcoma, squamous cell carcinoma, stomach cancer,thyroid cancer, topical skin lesion, veticulum cell sarcoma, Wilm'stumor, and/or combinations thereof.

In one embodiment, the graphene oxide (128) is functionalized withpolyethylene glycol. For example, expandable graphite may be exfoliatedand heated at about 900° C. for about 1 hour under argon to removeintercalated acid molecules. Then, a salt such as NaCl may be added andremoved by filtration with water to reduce particle size. Then, a strongacid, such as sulfuric acid, may be added to effect intercalation.Further, an oxidizing agent such as KMnO₄ may be added and the productmay be washed. Subsequently, carboxylic acid functional groups made beadded along with NaOH followed by sonication, neutralization, filteringand washing. The product formed is then typically a carboxylic acidmodified graphite oxide (GO—COOH). This product may be then sonicatedwith a 6-arm polyethylene glycol-amine andN-(3-dimethylaminopropyl-N′-ethylcarbodiimide) hydrochloride may beadded. Finally, mercaptoethanol may be added and the product subjectedto centrifugation in PBS to form NGO-PEG.

In still other embodiments, the graphene oxide (128) is functionalizedwith a linking molecule (such as linking molecule 36 described above atleast with reference to FIGS. 1B, 1C, and 1D), e.g. a linker such asGMBS which is known as N-[γ-maleimidobutyryloxy]succinimide ester in theart. The linking molecule (36) is not particularly limited. It is alsocontemplated that the graphene oxide (128) and/or the linking molecule(36) may be functionalized or bonded to a marker (such as the marker 38also described above at least with reference to FIGS. 1B, 1C, and 1D)such as a protein such as NeutrAvidin. See also, e.g., FIGS. 12A/B and14A/B. The protein may be directly bonded to the graphene oxide (128)and/or the linking molecule (36). It is further contemplated that thegraphene oxide (128), the linking molecule (36), and/or the marker (38)may be functionalized or bonded to an antibody (such as the antibody 40described above at least with reference to 1A-D) such as EpCAM againstthe EpCAM antigen expressed on the surface of cancer cells. The antibody(40) may be directly bonded to the graphene oxide (128), the linkingmolecule (36), and/or the marker (38). In one embodiment, the grapheneoxide (128) is disposed on a plurality of extensions (126), isfunctionalized with (i.e., bonded to) a linking molecule (36) which, inturn, is functionalized with (i.e., bonded to) a protein which, also inturn, is functionalized with (i.e., bonded to) an antibody (40). Theantibody (40) can then bind a rare cell (22) such as a CTC. The instantdisclosure is not limited to the aforementioned antibodies, proteins,etc. and one or more known in the art may be utilized and bonded to thegraphene oxide (128). Suitable non-limiting examples include variousantibodies and/or proteins, epithelial surface markers such as EGFR,prostate markers such as PSMA, PSA, cancer cell markers such as CD133,CD44, ALDH, endothelial markers such as CD31, CD34, leukocyte markerssuch as CD45, CD4, exosome/microvessicle markers such as CD63, etc.Alternatively, various peptides recognizing particular DNA sequences maybe utilized.

In one embodiment, the graphene oxide is functionalized with a bindingagent, the binding agent includes the reaction product ofphospholipid-polyethylene-glyco-amine (PL-PEG-NH₂) andN-γ-maleimidobutyryloxy succinimide ester (GMBS), the reaction productis further bonded to a protein, and the protein is further bonded to anantibody for interaction with the rare cells.

Additional Non-Limiting Embodiments of a Microfluidic Device:

In one embodiment, the system (120) is further defined as a microfluidicdevice. The microfluidic device (and/or system (120)) may include amicrofluidic channel (132) and/or a microfluidic chamber (156) throughwhich blood, body fluids, and/or other substances can flow. Typically,larger devices include microfluidic chambers (156) as opposed tomicrofluidic channels (132), but this is not necessarily true in everyembodiment. The microfluidic device (and/or system (120) and/ormicrofluidic channel (132) and/or microfluidic chamber (156)) may alsoinclude one, two, or a plurality of posts (e.g., extensions 126), suchas PDMS posts, to support one or more chambers, channels, or layers.

In one embodiment, the microfluidic device has one or more microfluidicchannels (132) and/or chambers (156) one or more of which eachindependently having a length, height, and/or width of from 1 μm to 1000μm (i.e., 1 mm) In various embodiments, one or more of these values isfrom 1 μm to 100, 5 to 95, 10 to 90, 15 to 85, 20 to 80, 25 to 75, 30 to70, 35 to 65, 40 to 60, 45 to 55, or 50 to 55, μm. In other embodiments,one or more of these values is from 100 to 1000, from 125 to 975, from150 to 950, from 175 to 925, from 200 to 900, from 225 to 875, from 250to 850, from 275 to 825, from 300 to 800, from 325 to 775, from 350 to750, from 375 to 725, from 400 to 700, from 425 to 675, from 450 to 650,from 475 to 625, from 500 to 600, from 525 to 575, or from 550 to 575μm. In other embodiments, the width may be up to 5 mm, and the length upto 100 to 1000 mm. The dimensions of the microfluidic device, as awhole, are not particularly limited. Any of the aforementioned valuesmay, for example, vary by 1, 2, 3, 4, 5, 10, 15, 20, or 25+% in varyingnon-limiting embodiments. All values, and ranges of values, between andincluding the aforementioned values are also hereby expresslycontemplated in various non-limiting embodiments.

One of more microfluidic channels (132) and/or chambers (156) may eachindividually have a unique shape and/or structure. In addition, onemicrofluidic channel (132) and/or chamber (156) may have a shape orpattern different from another microfluidic channel (132) and/or chamber(156) in the same device. The geometry of these patterns is also notparticularly limited. The patterns may be geometric, non-geometric,uniform or non-uniform, e.g. straight, zig-zag, herringbone, circular oroval, triangular, whorl-shaped, ribbon-shaped, marble, spiral-shaped,coil-shaped, curl-shaped, twisted, looped, helix, serpentine,sinusoidal, winding, and/or random, and the like. The shape or patternof the channels (132) and/or chambers (156) may be defined by the shapeand pattern of the spacing between adjacent extensions (126).

The microfluidic device typically utilizes radial flow for capture ofrare cells in a fluid, such as a bodily fluid. As previously describedat least with reference to FIGS. 27 and 28, the microfluidic device hasan inlet at the center axis (A) of the substrate (124) and at least oneoutlet at the outer edge (125) of the substrate (124). Fluid flows intothe microfluidic device through the inlet, flows through the channels(132) and/or chambers (156), and flows through the outlet(s) and leavesthe microfluidic device. The fluid generally flows radially from theinlet, through the channels (132) and/or chambers (156), and to theoutlet. However, as previously described, the channels (132) and/orchambers (156) are interconnected between the plurality of extensions(126). Accordingly, radial flow from the inlet towards the outlet of themicrofluidic device may not occur in a generally straight line in aradial direction from the inlet towards the outlet. Instead, flow fromthe inlet towards the outlet of the microfluidic device occurs inmultiple directions as the fluid flows through and between the multipleextensions (126) of the microfluidic device as the fluid heads radiallytoward the outlet of the microfluidic device. Examples of the flowprofiles of the fluid as the fluid heads radially toward the outlet ofthe microfluidic device are set forth in FIGS. 34A-D, 36, and 37.

In an embodiment, the microfluidic device includes a plurality ofbean-shaped extensions (126) with the concave side (133) of eachbean-shaped extension (126) facing toward the center axis (A) of thesubstrate (124). Further, each of the bean-shaped extensions (126) inthe second row (R₂) is typically offset (e.g., not entirely aligned)behind an extension (126) in the first row (R₁), and each of thebean-shaped extensions (126) in the third row (R₃) is typically offsetfrom an extension (126) in the second row (R₂), and so on. With thisorientation of the bean-shaped extensions (126) on the substrate (124),each bean-shaped extension (126) can intercept the movement of the fluidflowing from the center axis (A) toward the outer edge (125) of thesubstrate (124). When intercepted, the fluid directly contacts theconcave surface (135) of the bean-shaped extension (126), and flowsaround both of the lobes of the bean as the fluid continues to flowtoward the outer edge (125) of the substrate (124). An example of theflow profile of fluid flowing around the bean-shaped extensions (126) isshown in FIGS. 34C and D and FIG. 37.

The microfluidic device utilizing radial fluid flow provides a viablealternative to microfluidic devices which utilize linear fluid flow.Without being bound to any theory, it is believed that limitations existfor linear flow devices for effectively capturing rare cells at higherflow rates. It is believed that this is due, at least in part, to theconstant velocity of the fluid flow through the linear fluid flow device(as shown, e.g., in the flow profiles set forth in FIGS. 34A and B). Incontrast, radial flow overcomes these limitations in that the velocityof the fluid flowing through the device decreases with increasing crosssectional area (as shown, e.g., in FIG. 36), thereby providing varyingshear rates across the radius of the device. Accordingly, the rare cellswould experience different shear rates at every radius and would getcaptured at an optimal shear rate as determined by their surface antigenexpression.

Further, the concave surface (135) of the bean-shaped extensions (126)facing incoming flow provides a higher surface area for capture of therare cells. As shown in FIGS. 31A and B, for example, the rare cellcontacts and sticks to the concave surface (135) as the fluid flows byand contacts the extension (126).

The microfluidic device efficiently and effectively isolates rare cellsin one step at high flow rates (such as, e.g., a rate or velocity of upto about 10 mL/hr) using an affinity reaction between an epithelial celladhesion molecule (EpCAM) antigen expressed by rare cells, such as anepithelial tumor cell, and anti-EpCAM coated on the bean-shapedextensions (126) on the microfluidic device. At the high flow rate, rarecells may be captured through the radial flow device that providesvarying shear across the device for optimal capture conditions even atsuch high flow rate(s). Further, while the microfluidic device mayoperate at a flow rate of up to 10 mL/hr, it is to be understood thatthe microfluidic device may also operate at flow rates lower or higherthan 10 mL/hr.

The microfluidic device may also be designed to allow for optical orvisual inspection of the microfluidic channels (132) and/or microfluidicchambers (156). For example, the microfluidic device may include a top,bottom, and/or side, which may be transparent to allow for optical orvisual inspection. Alternatively, the microfluidic device may include atop, bottom, and/or side which may be opaque. It is also contemplatedthat the microfluidic device may not include a top.

The volume of the microfluidic channel (132) and/or microfluidic chamber(156) may be customized depending on a volume of the sample used. Thevolume of the microfluidic channel (132) and/or microfluidic chamber(156) may be smaller or larger than the size of the sample or may beapproximately the same as the size of the sample. In variousembodiments, the microfluidic device and/or the microfluidic channel(132) and/or microfluidic chamber (156) has a volume of from about 10 μLto 20 mL, from about 100 μL to 15 mL, from about 100 μL to 10 mL, fromabout 100 μL to 5 mL, from about 100 μL to 1 mL, or from about 100 μL to0.5 mL. However, these volumes are not limiting and the volume of themicrofluidic device and/or the microfluidic channel (132) and/ormicrofluidic chamber (156) may be greater or less than those describedabove. Any of the aforementioned values may, for example, vary by 1, 2,3, 4, 5, 10, 15, 20, or 25+% in varying non-limiting embodiments. Allvalues, and ranges of values, between and including the aforementionedvalues are also hereby expressly contemplated in various non-limitingembodiments.

The microfluidic channel (132) and/or microfluidic chambers (156) may bemodified to increase surface area, volume, etc. to increase aprobability that a rare cell with be captured. For example, when thewalls of the channels (132) may be substantially planar, the height ofthe microfluidic channels (132) and/or microfluidic chambers (156) maybe designed so that rare cells are more efficiently detected and/ortrapped.

The microfluidic device is not particularly limited to any particularefficiency. However, in various embodiments, the microfluidic device cantypically identify, enumerate, detect, capture, and/or isolate from 1 to10,000, 1 to 7,500, 1 to 5,000, 1 to 2,500, 1 to 1500, from 5 to 1000,from 10 to 500, from 25 to 200, or from 50 to 100, rare cells (22) froma blood sample of about 1 mL or less. Alternatively, the system (120)and/or microfluidic device may have a rare cell capture efficiency of atleast 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 99, percent determined as (rare cells captured on the(functionalized) graphene oxide (or any protein, antibody, marker, etc.bound thereto) divided by a total number of rare cells introduced to thesystem and/or microfluidic device) multiplied by 100. In otherembodiments, the system (120) and/or microfluidic device may have a rarecell capture efficiency of 95 to 100, 90 to 95, 90 to 100, 85 to 95, 85to 90, 80 to 85, 80 to 90, 80 to 95, 75 to 80, 75 to 85, 75 to 90, 75,to 95, 70 to 75, 70 to 80, 70 to 85, 70 to 90, 75 to 95, 50 to 95, 55 to90, 60 to 85, 65 to 80, 65 to 75, 65 to 70, 25 to 50, 59 to 75, or 25 to75, percent, as determined using the formula described immediatelyabove. In various embodiments, the microfluidic device has a rare cellcapture efficiency of about 70, 75, or 80 plus or minus about 20, 25, or30, at 5-20 cells/mL spiked in blood. Any of the aforementioned valuesmay, for example, vary by 1, 2, 3, 4, 5, 10, 15, 20, or 25+% in varyingnon-limiting embodiments. All values, and ranges of values, between andincluding the aforementioned values are also hereby expresslycontemplated in various non-limiting embodiments.

In various embodiments, the microfluidic device and system may capture,on average, about 155±236 CTCs/mL for NSCLC, about 16 to 292 CTCs/mL formetastatic prostate, about 25 to 174 CTCs/mL for localized prostatecancer, about 9 to 831 CTCs/mL for pancreatic cancer cells, about 5 to176 CTCs/mL for breast cancer cells, and about 42 to 375 (121±127)CTCs/mL for colorectal cancer cells. The microfluidic device may allowcaptured cells to be grown and cultivated and/or washed such thatnon-specifically bound cells, e.g., leukocytes, may be removed which mayresult in about a 10⁶-fold enrichment. Any of the aforementioned valuesmay, for example, vary by 1, 2, 3, 4, 5, 10, 15, 20, or 25+% in varyingnon-limiting embodiments. All values, and ranges of values, between andincluding the aforementioned values are also hereby expresslycontemplated in various non-limiting embodiments.

The microfluidic device may also include or be coupled to one or morecomponents such as reservoirs, pumps, valves, actuators, pipes, tubes,electrodes, meters, computers, electronic monitoring devices, analyticaldevices, electrical potential and/or resistance monitoring devices, andthe like. Those of skill in the art may select one or more of thecomponents to couple to the microfluidic device.

In one embodiment, the microfluidic device includes a silicon substratethat includes approximately has 60,000 extensions (e.g. formed fromgold) disposed in bean-shaped patterns. The distance between eachextension in a row (R) or between rows (R), in this embodiment, is about25 to 32 μm. Graphene oxide nanosheets may be self-assembled onto theextensions (126), in this embodiment, at a thickness of about 100 nm.The graphene oxide of this embodiment may also be functionalized withEpCAM antibodies. It is contemplated that the aforementioneddescriptions and characteristics are not necessarily limited to thisparticular embodiment and may apply to other embodiments describedherein.

Method for Forming the System (20), (120) and/or Microfluidic Device:

This disclosure also provides a method of forming the system (20), (120)and/or a microfluidic device. The method typically includes the steps ofproviding the substrate (24), (124), providing the extension (26),(126), and providing the (functionalized) graphene oxide. The substrate(24), (124) may be provided as a single layer or as more than one layer,including one or more supplemental layers (34). Accordingly, the methodmay include the step of forming the substrate (24), (124) including oneor more layers. The step of forming the substrate (24), (124) and/or theone or more supplemental layers (34) is not particularly limited, may beany known in the art, and may be as described above.

The step of providing the extension (26), (126) is also not particularlylimited and may include any method of forming and/or depositing theextension (26), (126) on the substrate (24), (124). For example, themethod may include the step of evaporating and patterning metal (e.g.Cr/Au) layers. Alternatively, the method may include the step of etchingsilicon to form the extension (26), (126).

The step of providing the (functionalized) graphene oxide again is notparticularly limited. The step of providing may include, or be furtherdefined as, forming the graphene oxide, and/or reacting the grapheneoxide to functionalize the graphene oxide with one or more markers,proteins, etc. as described above. The graphene oxide may be formedand/or reacted or functionalized by any method or reaction known in theart, including those reactions and functionalization reactions describedabove. Each of the aforementioned substrate (24), (124), extensions(26), (126), and graphene oxide (28), (128) may be assembled together toform the system and/or microfluidic device using any method known in theart.

Method for Detecting Rare Cells:

This disclosure also provides a method for detecting rare cells usingthe system (20), (120) and/or microfluidic device of this disclosure.The method allows for small amounts of bodily fluid to be evaluatedaccuracy and precisely and in a time and cost effective manner todetermine the presence of rare cells.

The method includes the steps of providing the system (20), (120) and/ormicrofluidic device and introducing a sample of bodily fluid to thesystem (20), (128) and/or microfluidic device such that the sampleinteracts with the (functionalized) graphene oxide (28), (128) and/orany proteins, markers, antibodies, etc. bonded thereto. The methodallows for small amounts of bodily fluid to be evaluated accuracy andprecisely and in a time and cost effective manner to determine thepresence of rare cells (22). The step of providing the system (20),(120) and/or microfluidic device is not particularly limited and mayinclude one or more of the aforementioned steps described as associatedwith the method of forming the system (20), (120) and/or themicrofluidic device.

The step of introducing a sample of bodily fluid is also notparticularly limited. Typically, this step is further defined asexposing the system (20), (120) and/or the microfluidic device and/orthe extension (26), (126) to the bodily fluid such that the bodily fluidcontacts the extension (26), (126) and the (functionalized) grapheneoxide (28), (128), which is typically modified or functionalized in suchas a way as to interact with the bodily fluid in a designated manner. Inone embodiment, the step of introducing the bodily fluid is furtherdefined as injecting or adding the bodily fluid to the entrance or inletof the microfluidic device. The method may also include the step offlowing the bodily fluid through the microfluidic channel(s) (132)and/or microfluidic chamber(s) (56), (156). For the system (20), thestep of flowing the bodily fluid through the channel (32) and/or chamber(56) occurs, e.g. along the longitudinal axis (L₁), from the upstreamend (46) towards the downstream end (48) and out of the exit (52). Thesteps of this method may include any of the parameters and/ordescriptions associated with the system, as described above.

Another embodiment of the method for detecting rare cells in the fluidutilizes the system (120). In this embodiment, the method includesintroducing a sample of fluid containing the rare cells into the inletof the system (120) such that the sample of fluid flow radially from theinlet toward the outer edge (125) of the substrate. In an embodiment,the step of introducing the sample is accomplished at a rate of up toabout 10 mL/hr. Furthermore, the rate decreases as the fluid flows fromthe inlet of the system (120) towards the outer edge (125) of thesubstrate (124). The method further includes capturing the rare cells asthe rare cells interact with the functionalized graphene oxide disposedon the extensions (126).

Method for Diagnosing Cancer:

This disclosure also provides a method for diagnosing a cancer orcarcinoma in a subject. This method includes the step of introducing asample of a bodily fluid to the system (20), (120) and determiningwhether any target rare cells (22) are present. Rare cells (22) obtainedby the methods of the disclosure may be assayed for genetic information.In addition, the rare cells (22) may be assayed for changes in geneticinformation over time as well as or in the alternative to enumeration,e.g. to monitor for the appearance of mutations that indicate a changein therapy is advisable.

Method for Lysing Rare Cells:

This disclosure further provides a method of lysing rare cells (22)using the system (20), (120) of this disclosure. This method typicallyincludes the step of introducing a sample of a bodily fluid to thesystem (20), (120) and subsequently introducing a lysing agent to thesystem. The lysing agent may be any known in the art.

One or more methods of this disclosure may also include the step ofwashing the rare cells (22) at a high shear stress or volume to increasepurity and reduce the number of weakly bound or non-specifically boundrare cells (22) in the system (20), (120) and/or microfluidic device.One or more methods of this disclosure may also include the step ofcounting or quantifying a number of bound rare cells (22). The rarecells (22) can be counted by any method known in the art, includingoptical, e.g. visual inspection, automated counting, microscopy baseddetection, FACS, and electrical detection, e.g. with the use of Coultercounters. Counting of the rare cells (22) can be useful for diagnosingdiseases, monitoring the progress of disease, and monitoring ordetermining the efficacy of a treatment. The number of rare cells (22)may also be counted in non-medical applications, e.g. for determinationof the amount, presence, or type of contaminants in environmentalsamples, pharmaceuticals, food, or cosmetics.

One or more of the methods of this disclosure may also include the stepof measuring a desired characteristic of rare cells (22). For example,the method may include the step of measuring desired biologicalproperties of rare cells (22) such as mRNA expression, proteinexpression, and DNA quantification.

It is also contemplated that the disclosure may include one or moreelements, one or more methods, one or more devices, and/or one or moresystems as described in one or more of the following references, each ofwhich is expressly incorporated herein by reference, in one or morenon-limiting embodiments: Y. Shao, J. Wang, H. Wu, J. Liu, I. A. Aksay,Y. Lin, “Graphene Based Electrochemical Sensors and Biosensors: AReview,” Electroanalysis, Vol 22, pp. 1027-1036, 2010; Y. Liu, D. Yu, C.Zeng, Z. Miao, L. Dai, “Biocompatible Graphene Oxide-Based GlucoseBiosensors,” Langmuir, vol. 26, pp. 6158-6160, 2010; J. H. Jung et al.,“A Graphene Oxide Based Immuno-biosensor for Pathogen Detection,”Angewandte Chemie, vol. 122, pp. 5844-5847, 2010.

The instant disclosure may also include one or more elements, one ormore methods, one or more devices, and/or one or more systems asdescribed in the provisional application (U.S. Prov. App. Ser. No.61/541,814) and/or appendix filed therewith. It is to be very clear andunderstood that nothing in any one or more of any reference cited hereinand incorporated by reference herein, or in the appendix, is to limitthis disclosure. The various embodiments and options described in thereferences, provisional patent application, and appendix which areincorporated herein by reference are not limiting, are optional, and arein no way meant to limit this disclosure.

EXAMPLES Simulation of One Embodiment of a Pattern of Extensions in aMicrofluidic Device

A simulation of fluid flow in a theoretical microfluidic device isperformed with Comsol Multiphysics 4.2. A three-dimensional leaf patternof (gold) extensions disposed on a substrate is generated and analyzedwith a laminar flow module, the results of which are set forth in FIGS.6 and 7. The height of the extensions is assumed to be 3 μm although theactual height of the gold is about 100 nm. An effective height of theextensions including the gold may be as high as 1-3 μm because ofaddition of functional groups.

In the simulation, the effect of the leaf pattern on a fluid field isexamined by increasing a height of the fluid field from 10 μm to 30 μm,see FIG. 6. The Figs. show that, at or below 10 μm, the fluid field isdisturbed significantly by the leaf pattern while, as the height of theleaf pattern increases, the effect of this disturbance decreases. Thisdisturbance may enhance the cell-surface interactions thus increase thecapture efficiency.

To evaluate the performance of the leaf pattern, the simulation on acircular pattern is conducted as set forth in FIGS. 7. As shown in bothFIGS. 6 and 7, the simulation demonstrates that the leaf patternoutperforms the circular pattern by increasing the number of boundarylayers thereby enhancing particle-surface interactions. This effect iswell illustrated at 10 μm height of a channel of a microfluidic device.

Example of One Embodiment of the System/Microfluidic Device of thisDisclosure:

In one embodiment, the system/microfluidic device of this disclosureutilizes a flower shaped architecture for the extensions, e.g. as setforth in FIGS. 2G and 4C, disposed on a silicon substrate. The siliconsubstrate of this example has 58,957 flower-shaped gold extensions eachhaving an approximate height and width of 100 μm×100 μm. The distancebetween each extension in a column is 150 μm. The overall size of thesystem/microfluidic device is 24.5 mm×60 mm×3 mm A PDMS layer of thesystem/microfluidic device has a microfluidic chamber with 50 μm heightand a 45 μL volume. Graphene oxide nanosheets are self-assembled ontothe patterned gold extensions. The extensions including the grapheneoxide are chemically functionalized with EpCAM antibodies. The effectivefunctionalized surface area enables the system/microfluidic device to bea simple polydimethylsiloxane (PDMS) flat chamber-like structure.

More specifically, the graphene oxide of this example is non-covalentlyfunctionalized with phospholipids-polyethylene-glyco-amine (PL-PEG-NH2).The hydrophobic lipid chains of PL-PEG-NH2 are immobilized onto thesurface of the graphene oxide. The functionalized graphene oxide has ahigh water solubility, biocompatibility, and functional groups availablefor further bioconjugation. Tetrabutylammonium (TBA) hydroxide is addedfor intercalation and complete exfoliation of graphene oxide. TBAcations and the amino group of PL-PEG-NH2 can interact with the goldsurface by electrostatic attraction. N-γ-maleimidobutyryloxy succinimideester (GMBS) has N-hydroxysuccinimide (NHS) esters that react with aminegroups of graphene oxide-PEG to form amide bonds.

In this example, CTCs are captured by using the following NeutrAvidinand biotinylated EpCAM antibody interactions. To functionalize andself-assemble graphene oxides before bonding, the silicon substrate isdipped into a functionalized graphene oxide suspension and grapheneoxide self-assembles on the gold surface of the extensions. SEM imagesreveal that gold patterns are covered with functionalized grapheneoxides, see e.g. FIG. 11C. The functionalized graphene oxides areattached to the sides of the extensions as well as onto the top of theextensions. This phenomenon demonstrates the high selectivity ofgraphene oxide assembly on the gold extensions rather than onto thesilicon substrate and the uniform assembly and saturation density ofgraphene oxide on the gold extensions. AFM images, as set forth in FIG.11, show graphene oxide sheets are disposed on the gold extensions. Thethickness of the graphene oxide in this example is about 1-3 nm.

To confirm that the graphene oxide is functionalized, fluorescentlylabeled NeutrAvidin is utilized, see e.g. FIGS. 12A and 14A/B. Goldextensions including modified graphene oxide specifically show thepresence of fluorescence, see e.g. FIG. 12A, where as negative controlswithout NeutrAvidin show no fluorescence at the same exposure condition,see e.g. FIG. 12B. This indicates high sensitivity and specificity,which is expected to exhibit high cell capture efficiency when employedto isolate viable cancer cells from whole blood samples.

To characterize the system/microfluidic device, MCF-7 cells are labeledwith fluorescent cell tracker dye and spiked into buffer at variousconcentrations and flowed through the system/microfluidic device at adesired flow rate, e.g. 1,000 cell/mL, 1 mL/hr, as described below.Subsequently, the captured cells are counted.

More specifically, 100-1000 MCF-7 cells are spiked into the buffersolution. Samples of the spiked buffer solution are then flowed throughthe system/microfluidic device at various rates, see e.g. FIGS. 18 and19. At a flow rate of 1-3 mL/hr, the capture yield is over 80%. At a 1mL/hr flow rate, a very small difference of the capture yield betweenflow rates is observed, see e.g. FIGS. 13A-G.

A comparative silicon device that does not include the extensions or thefunctionalized graphene oxide is also produced. More specifically, asilicon substrate is functionalized with silane, GMBS, and NeutrAvidinusing the same conditions as are used to form the example of thesystem/microfluidic device of this disclosure. Samples of the buffersolution are then passed over this comparative silicon device todetermine how many cells are captured. The results of this experimentare set forth in FIG. 21.

These results demonstrate that use of the graphene oxide and theextensions increase the capture efficiency likely due to enhancedsurface area and morphology. When MCF-7 cells are flowed through theinstant system/microfluidic device, the trajectory of cells tends to bea parabola. Along with the trajectory, most of cells are captured onextensions, see e.g. FIGS. 13A-G.

To further investigate the capture efficiency using blood samples, a lownumber of MCF-7 cells (3-5 cells, 10-20 cells, 100 cells, see e.g. FIG.17A) are spiked into 1 mL of whole blood (FIG. 20). Cells are diluted inserum-free medium at a concentration of 1×10⁵ cells/mL. 1 μL of cellsuspension added to a 96-well plate. The transferred cells are countedunder a microscope, immediately transferred by pipette and added to 1 mLof whole blood. Remaining cells not transferred into the whole blood arealso counted to verify the accuracy of the transfer. By subtracting thenumber of remaining cells from the original count, an actual number ofcells spiked into blood is estimated. These steps occur at roomtemperature.

The average recovery rates of the 10-20 cell spike and the 100 cellspike are 91% and 87%, respectively. The average recovery rate of the3-5 cell spike is 59%. Two examples that include 6 cells exhibit a 100%recovery rate

Microfabrication of the Microfluidic Device:

The aforementioned system/microfluidic device is fabricated using aseries of 4 inch N-type silicon wafers as substrates. These wafers arecleaned by RCA cleaning. A 3000 Å thermal oxide is grown by a wetoxidation process. Subsequently, Cr and Au (100 Å/1000 Å) layers aredeposited on the silicon wafer by e-beam evaporation to form extensions.Then, a photoresist is coated by automatic spinner and patterned by maskaligner (MA-6, Karl Suss). For patterning the Au and Cr extensions, thewafers are placed in Au/Cr etch solution. The photoresist is thenremoved by acetone and rinsed by isopropyl alcohol (IPA).

Subsequently, an already prepared PDMS layer with a chamber having a 50mm length, an 18 mm width, and a 50 μm height, is bonded onto thesilicon wafer having the pattern of gold extensions disposed thereon.The PDMS layer is fabricated using standard SU-8 mold process. Thesilicon wafer and the PDMS layer are bonded by corona dischargetreatment.

Functionalization and Nano-Assembly of Graphene Oxide:

10 mg of graphene oxide (Cheap Tubes Inc.) powder is also prepared toform a single layer graphene oxide (SLGO) which is then treated with amodified Hummer's method as described in Hummers, W. S. & Offeman, R. E.Preparation of Graphitic Oxide. Journal of the American Chemical Society80, 1339 (1958). 10 mL N,N-dimethylformamide (DMF) and 300tetrabutylammonium (TB A) hydroxide (40% in water) are then addedthereto to form a graphene oxide suspension, see e.g. FIG. 16A. Using atip sonicator, the graphene oxide suspension is then ultrasonicated for30 min. To avoid an increased temperature during the sonication, atemperature sensor is monitored and the suspension tube is immersed inan ice bath. The graphene oxide suspension is reserved for 3 days atroom temperature.

4 mL of the supernatant is then extracted and 15 mg ofphospholipids-polyethylene-glyco-amine (PL-PEG-NH2, NOF Co.) isdissolved therein, bath sonicated for about 1 hour, and subsequentlycentrifuged at 12,000 rpm for 3 min. The supernatant is collected andstored at 4° C. The supernatant includes PEG functionalized grapheneoxide. The silicon wafer is then dipped into the functionalizedsupernatant for 10 min and washed with DI water and IPA, see e.g. FIG.11A/B. The prepared PDMS layer with the chamber, as described above, isthen bonded to the silicon wafer using corona discharge treatment.

A GMBS solution is then flowed through the chamber at 20 μL/min using asyringe pump (Harvard Apparatus). After 30 minutes of incubation, thechamber is washed with ethanol at 100 μL/min Subsequently, 50 μg/mLNeutrAvidin is prepared and flowed through the chamber at 20 μL/minAfter 1 hour incubation, the chamber is flushed with phosphate bufferedsolution (PBS) at 100 μL/min to remove excess NeutrAvidin. Finally,biotinylated EpCAM antibody at a concentration of 20 μg/mL in PBS with1% (w/v) BSA is flowed through the chamber for 10 minutes at 20 μL/minAfter 1 hour incubation, PBS is flowed through the chamber to wash, andthen, bovine serum albumin (BSA) is added to a 1×PBS solution and flowedthrough the chamber at 100 μL/min for 5 minutes. After flowing BSAsolution, the chamber incubates for 30 minutes.

Cell Culture and Labeling:

Tissue culture reagents are purchased from GIBCO InvitrogenCorporation/Life Technologies Life Sciences unless otherwise specified.MCF-7 and PC-3 cells are cultured in DMEM and DMEM/F12 medium including10% fetal bovine serum and 1% penicillin-streptomycin solution. Whencells reach more than 90% confluence, the medium is replaced with afresh medium. A green cell tracker dye (Invitrogen, CellTracker GreenCMFDA, C7025) for labeling cells is used to perform rare cell captureefficiency calculations.

Blood Specimen Collection:

Blood samples are drawn from patients with tumors and healthy donorsafter obtaining informed consent under an IRB-approved protocol. Allspecimens are collected into EDTA tubes and are processed within 3hours.

Cytokeratin and CD45 Staining:

After incubating for 30 minutes, 1 ml blood samples includingnon-labeled cells are flowed through the chamber at a rate of 1 ml/hr tocapture cells. Subsequently, the captured cells are washed with PBS,fixed with 4% paraformaldehyde (PFA), permeabilized with 0.2% Triton-Xand incubated for 30 minutes followed by washing with PBS. The chamberis then incubated for 30 minutes with 1 mL of blocking buffer including2% normal goat serum and 3% BSA. Anti-cytokeratin (BD Biosciences) andanti-CD45 (BD Biosciences) are diluted at 1:50 and 1:10 in 1% BSA,respectively, see e.g. FIG. 17A. Antibodies are then flowed through thechamber for 20 minutes at 50 μL/min and incubated for 1 hour. Afterabsorption of the primary antibody, the chamber is washed with PBS. Theanti-cytokeratin is probed with AlexaFluor488 IgG2a FITC (Invitrogen)and the anti-CD45 is probed with AlexaFluor546 IgG1 (Invitrogen). Thesecondary antibodies are diluted in 1% BSA at a 1:1000 ratio, flowedthrough the chamber for 20 minutes at 50 μL/min, incubated for 1 hourand then washed with PBS.

To stain nuclei of captured cells, DAPI (1:1000 dilution in PBS) isflowed through the chamber for 20 minutes at 50 μL/min and the chamberis incubated for 15 minutes and washed with PBS.

Cell Treatment with EdU

To measure cells' ability to proliferate, Click-iT EdU Imaging Kit(Invitrogen, C10340) is used. After capturing cells, the chamber iswashed with PBS and 1 μM EdU is added to the chamber. The chamber isincubated overnight, washed with PBS, and then the cells are fixatedusing 4% PFA. After 15 min incubation, the chamber is washed with 3% BSAtwice, followed by cell permeabilization with 0.2% Triton X-100 in PBSand incubated for 20 minutes. The chamber is washed with 3% BSA twiceand 0.5 mL of Click-iT EdU buffer additive is added, followed byincubation for 30 minutes and washing with 3% BSA. For nucleus staining,1 mL of 1× Hoechst 33342 solution is added and cells in the chamber areincubated for 30 minutes and washed with 1 mL of PBS.

Additional Examples

Various solutions of solvent and functionalized graphene oxide sheetsare formulated and exposed to various metals that are representative ofvarious extensions of the system of this disclosure. The metals are thenanalyzed to determine whether any of the metals are able to adsorb thefunctionalized graphene sheets. The details of these analyses are setforth immediately below in Table 1. The results associated with theseevaluations are determined using scanning electron microscopy and/oratomic force microscopy.

TABLE 1 Functionalized Graphene Metal Oxide Sheet Solvent Result Au TBA,PL-PEG-amine H₂O/DMF (3:100) Positive Au TBA, PL-mPEG H₂O/DMF (3:100)Positive Au PL-PEG-amine H₂O/DMF (3:100) Negative Au TBA, PL-PEG-aminePure DMF Negative Au TBA, PL-PEG-amine, H₂O/DMF (3:100) Negativeacidified by HNO₃ Al TBA, PL-PEG-amine H₂O/DMF (3:100) Negative Ti TBA,PL-PEG-amine H₂O/DMF (3:100) Partially Positive Co TBA, PL-PEG-amineH₂O/DMF (3:100) Partially Positive Pd TBA, PL-PEG-amine H₂O/DMF (3:100)Partially Positive

Additional non-limiting examples of graphene oxide sheets andnon-limiting examples of this disclosure are also formed. In a firstexample, single layer graphene oxide (SLGO) powder is used and subjectedto a modified hummer's method as described in W. Hummers, and R.Offeman, “Preparation of graphitic oxide,” Journal of the AmericanChemical Society, vol. 80, pp. 1339, March 1958. More specifically, 10mg of graphene oxide powder is prepared. Then, 10 mL DMF and 300 μL TBAhydroxide (40%) is added thereto to form a solution. Using a tipsonicator, the solution is sonicated for approximately 10 min at anamplitude of 50 with a 2 minute cool down. A supernatant is then removedand reserved for 2-3 days. Approximately 4 mL of the supernatant is thenextracted and approximately 15 mg of PL-PEG-NH2 is dissolved therein.This solution is then bath sonicated for about 1 hour and subsequentlycentrifuged at 12,000 rpm for about 3 min. After centrifugation, thesupernatant is collected at stored at about 4° C. The supernatantincludes polyethylene glycol (PEG) functionalized graphene oxide.

To verify the functionalization of the graphene oxide with thepolyethylene glycol, the supernatant is analyzed using FT-IRspectroscopy. The spectra generated are then compared to similar FT-IRspectra of non-functionalized graphene oxide. The results of these FT-IRanalyses are set forth in FIGS. 15A and B. FIG. 15A shows that, at 3400cm⁻¹, there is a graphene oxide-related peak clearly shown. FIG. 15Bshows that, at 1650 cm⁻¹, the a peak for functionalized graphene oxidedisappears which is indicative of an NH—CO stretching vibration andexchange of an amine group with polyethylene glycol.

A microfluidic device, similar to the microfluidic device set forth inFIG. 1, is also formed. More specifically, the microfluidic deviceincludes a silicon substrate, an SiO₂ supplemental layer disposed on andin direct contact with the silicon substrate, an additional PDMSsupplemental layer, and gold nanoposts (as the extensions of thisdisclosure). The PDMS supplemental layer is fabricated using a standardSU-8 mold. The silicon substrate and the PDMS supplemental layer arebonded by oxygen plasma treatment. FIG. 5 shows steps in the fabricationprocess of this particular non-limiting microfluidic device. Morespecifically, an N-type (100) silicon wafer is cleaned by RCA cleaning.A silicon dioxide layer (3000 Å) is then grown by wet oxidationprocessing in a furnace at about 1100° C. Cr and Au (100 Å/1000 Å)layers are then deposited by e-beam evaporation. Photoresist (SPR 330)is then coated by an automatic spinner (ACS 200) and patterned by a maskaligner (MA-6). The exposure time is about 12 sec and the developingtime is about 25 sec for these processes. For patterning the Au and Cr,the wafer is placed in an Au etch solution for about 25 sec and into aCr etch solution for about 25 sec. The photoresist is then removed byacetone and rinsed by IPA and the device is exposed to oxygen plasma forabout 3 minutes at 100 W.

After formation of the microfluidic device and the functionalizedgraphene oxide described above, the PEG-functionalized graphene oxide isthen flowed through the microfluidic device. By self-assembly, thefunctionalized graphene oxide molecules are directed to bridge arrays ofthe gold nanoposts on the silicon substrate which helps remove anyextraneous coatings on the graphene oxide molecules and increases theelectrical conductance of the microfluidic device (e.g. through anelectrical annealing method). As a result of flowing the functionalizedgraphene oxide molecules through the microfluidic device, thefunctionalized graphene oxide molecules attach to the sides and tops ofthe gold nanoposts, as shown in FIG. 8, which is demonstrative of highselectivity of the deposition of functionalized graphene oxide moleculeson the gold nanoposts as opposed to the SiO₂ supplemental layer and/orthe PDMS supplemental layer. In addition, atomic force microscopy (AFM)is also utilized to characterize graphene oxide molecules disposed onthe gold nanoposts. As set forth in FIGS. 9A and 9B, an average size ofthe functionalized graphene oxide molecules disposed on the goldnanoposts is on a nanometer scale and the approximate topographic heightis about 1 to 1.5 nm. FIGS. 10A and 10B illustrate that little, if any,disposition of the functionalized graphene oxide on the gold nanopostsoccurs if no TBA is used, as described above. FIGS. 10C-10E illustratethe there is plentiful disposition of the functionalized graphene oxideon gold nanoposts if TBA is used.

Subsequently, the supernatant (i.e., a solution of the PEGfunctionalized graphene oxide) is flowed through the microfluidic deviceat about 10 μL/min for 20 minutes. The microfluidic device is thenwashed using DI water and IPA at 100-300 μL/min for 10 minutes,respectively. FIGS. 11A and 11B illustrate that the washing with IPA andDI water significantly cleans the gold nanoposts of excessfunctionalized graphene oxide, which is desirable to increase theselectivity of the device to the targeted rare cells. The PEGfunctionalized graphene oxide disposed on the gold nanoposts is thenfurther modified with fluorescently labeled NeutrAvidin to immobilizethe biotinylated epithelial-cell adhesion molecule antibody(anti-EpCAM). EpCAM is a transmembrane glycoprotein that is frequentlyoverexpressed in a variety of solid-tumor cells and is absent fromhematologic cells. More specifically, a GMBS linker is prepared in aglove box and flowed through the microfluidic device at 10 μL/min for 30minutes. The GMBS linker binds to the functionalized graphene oxide.Then, the microfluidic device is washed with Ethanol at 100 μL/min forabout 15 min Subsequently, the fluorescently labeled NeutrAvidin isapplied to the microfluidic device at about 10 μL/min for 30 minutes andbinds to the GMBS linker. As set forth in FIG. 12, fluorescencemicroscopy at varying exposure times and varying magnificationsdemonstrates that the fluorescently labeled NeutrAvidin successfullybinds to the GMBS linker and, in turn, to the functionalized grapheneoxide.

Then, the microfluidic device is washed using PBS at 100 μL/min for 10minutes. Then, EpCAM, a CTC antibody, is flowed through the microfluidicdevice at 10 μL/min for two 30 minute intervals and binds to thefluorescently labeled NeutrAvidin. After washing the microfluidic devicewith PBS at 100 μL/min for about 10 minutes, a 1% BSA solution is flowedthrough the microfluidic device at 100 μL/min for about 10 minutes.

To further test the capture of actual cancer cells, a buffer solution offluorescently labeled breast cancer cells (MCF-7) is formed wherein thecancer cell are present in a concentration of about 5,000 cells/mL. Thebuffer solution is then flowed through the microfluidic device at 10μL/min for about 50 minutes. Finally, PBS and PFA are flowed through themicrofluidic device at 100 μL/min for about 5 minutes, respectively.

Subsequently, the device is analyzed using fluorescence microscopy todetermine whether any of the of fluorescently labeled breast cancercells were captured by the EpCAM antibody. FIG. 13 illustrate thatfluorescence is observed which is indicative that the fluorescentlylabeled breast cancer cells are captured by the device, and morespecifically, through the use of the functionalized graphene oxide. Theresults also suggest that a high surface-to-volume ratio of thefunctionalized graphene oxide disposed on the gold nanoposts cangenerate 3D electrical surfaces that can significantly enhance detectionlimits and allow for highly reproducible detection of clinicallyimportant cancer markers.

Comparative Example

A control microfluidic device is also prepared using the same procedureas outlined above and washed with PBS except that no functionalizedgraphene oxide is utilized. In this control microfluidic device, thesame fluorescently labeled NeutrAvidin is utilized at the same rates andamounts as described above. However, since there is no functionalizedgraphene oxide present, the fluorescently labeled NeutrAvidin does notbind. To verify that the fluorescently labeled NeutrAvidin does not bindto the nanoposts in the comparative example, the comparative device isexamined using fluorescence microscopy, as described above. The resultsare set forth in FIG. 14A/B and demonstrate that, at exposure times ofeven up to 1 second, no fluorescently labeled NeutrAvidin is seen. Theseresult further highlight the sensitivity and specificity of thefunctionalized graphene oxide.

The results set forth above demonstrate the successful formation of anintegrated nano microfluidic device with functionalized graphene oxideand gold using an orthogonal integrated translational approach andbioengineering tools to identify and bind CTCs of a breast cancer cellline as a model system. The aforementioned non-limiting approachutilizes self assembly of graphene oxide in a unique way for enhancedsensitivity and specificity as a detection tool. Graphene oxide hasunique properties, such as increased 2D and 3D electrical conductivity,large surface area, superb mechanical flexibility, and increasedchemical and thermal stability. The chemically derived and noncovalentlyfunctionalized graphene oxide described immediately above has theability to overcome the limitations of carbon nanotubes (CNTs) such asvariations in electrical properties of CNT-based devices and the limitedsurface area of CNTs.

Example of Another Embodiment of the System (120)/Microfluidic Device:

The design of an optimal capture platform with structures relies largelyon channelizing the flow in order to have maximum cell contact withfunctionalized post surfaces. Reducing the flow separation is one way toincrease the contact of the cell with the post surfaces, therebyincreasing the chance of capture. A circular micropost as in the CTCchip(microfluidic device with linear flow) shows a large flow separationbehind the post and the area behind the post remains largely unused forcapture. To overcome this, it was believed that a bean-shape wouldprovide a better flow utilization for capture. The flow dynamics of aseries of different designs of bean-shapes that varied by arc angles andlength, followed by a qualitative estimate of boundary layer thicknessfrom fluid simulations was performed using COMSOL Multiphysics 4.2software. The OncoBean Chip (microfluidic device with radial flow) wasdesigned with bean-shaped posts conceived from an arc angle of 90°, withthe structures measuring 50 μm wide and 118 μm along the longest axis.The posts were placed 25-32 μm apart in polar arrays, with subsequentarrays being rotated to introduce interjection of flow. FIGS. 27, 34C,and 35 demonstrate velocity magnitude and shear rate profiles onsimulated sections. For a flow rate of 10 mL/hr, the simulationspredicted maximum velocity (0.0158 m/s) for the simulated inletdimensions and a decreasing trend on moving radially outward as thecross sectional area increased. Similar to the velocity profile, theshear rate decreased on moving toward the outlets. This continuousdecrease in shear even at a high flow rate of 10 mL/hr makes the radialflow device usable to capture cells with a heterogeneous expression ofantigens.

A particle trajectory was also simulated to predict hydrodynamicefficiency and to observe streamline paths. The plot in FIG. 37 shows 15μm rigid particles and corresponding streamlines as the particlesnavigate around the post structures (walls or extensions). Thesimulation showed that 94.3% of particles (33 of 35 particles sent)interact with bean posts within the first 600 μm array of dense posts,with the remaining 2 particles being interjected at other post regions.Accordingly, the simulation indicated a high hydrodynamic efficiency anda good streamline trajectory with sufficient cell-post interaction for asensitive capture.

Optimization of the System (120) for Cancer Cell Capture:

The OncoBean Chip was optimized for capture with the epithelial lungcancer cell line H1650 with anti-EpCAM as the capture antibody. Captureefficiency was calculated as the percentage of captured cells on thedevice to the total number of cells sent, normalized against theOncoBean Chip 1 mL/hr to account for cell spiking errors. Briefly,fluorescently labeled H1650 cells were spiked into healthy blood at aconcentration of 1000 cells mL⁻¹. To validate the effectiveness of theOncoBean Chip against a standard affinity-based microfluidic device, theOncoBean Chip was compared with a PDMS based version of the CTC chip.The following conditions were analyzed: OncoBean Chip 1 mL/hr, OncoBeanChip 10 mL/hr, CTC Chip 1 mL/hr and CTC Chip 10 mL/hr.

Upon comparison of performance with the CTC Chip, the OncoBean Chipshowed high capture efficiencies with mean yields of 100% and 82.7% atboth 1 mL/hr (n=4) and 10 mL/hr (n=4) respectively (see FIG. 38), inclose agreement with the mean capture yield of 90.7% obtained in the CTCChip at 1 mL/hr. Alternatively, the high capture rate achieved by theCTC Chip at 1 mL/hr dropped to a mean rate of 27.8% with the same chipat a flow rate of 10 mL/hr (n=3). This greater than 3-fold drop can beexplained by the linear flow profile in the CTC Chip, which may not beconducive for capture at high flow rates due to the high velocitiespresent constantly throughout the device. The OncoBean Chip combats thisby the radial flow design, as there is a continuous drop in the velocityon moving outward.

The effect of flow rates on the capture efficiency in the OncoBean Chipwas tested under four conditions: 1 mL/hr, 2.5 mL/hr, 5 mL/hr, and 10mL/hr on anti-EpCAM coated devices to test the robustness of the captureplatform at different velocities. As seen in FIG. 40, the captureefficiency had no significant drop on increasing the flow rate from 1 to10 mL/hr, with the mean capture yield being greater than 80% at all theabove flow rates. The lowest mean capture efficiency was 89.5% at 10mL/hr indicating a high yield even at the maximum flow rate.

A similar trend in efficiency was observed in the absence of othercells, as is seen in the capture of H1650 cells spiked into serum freemedium. The OncoBean Chip recovery was also tested with the breastcancer cell line MCF7, and the device achieved a mean capture yield of98% at 10 mL/hr normalized against the same device at 1 mL/hr at a spikeconcentration of 1000 cells mL⁻¹. This data suggests that it is possibleto obtain similar recovery of CTCs with a process that is 5-10 timesfaster than many standard affinity isolation methods using whole blood.

Purity:

Non-specific background blood cells may be a hindrance during molecularanalysis of CTCs, and effective CTC isolation as well as minimalcontamination of the background cells is important. The purity ofcapture in the OncoBean Chip was determined by measuring the number ofwhite blood cells (WBCs) per mL of whole blood captured on the device.Due to variations in WBC counts between different donors, a puritypercentage was not calculated; a relative comparison caused by increasein flow rates was performed. As shown in FIG. 39, the high flow ratestend to have an advantageous effect on reducing the non-specific captureof contaminating WBCs, with a 2-fold drop in the WBC count at 10 mL/hrcompared with 1 mL/hr (OncoBean Chip: 1 mL/hr range 1060-1240 cells permL, 10 mL/hr range 390-740 cells per mL, CTC Chip: 1 mL/hr range930-2010 cells per mL, 10 mL/hr range 330-850 cells per mL). This may beattributed to the high velocities at high flow rates which reduce theresidence time for binding of these cells. The nonspecific cell numbersshow large variation in capture between the OncoBean and CTC Chips, butthe nonspecific cell numbers indicate similar trends between the flowrates suggesting that the radial gradation in shear does not increasenon-specific white blood cell capture.

Cell Viability at High Flow Rates:

For optimal use of microfluidic platforms to capture CTCs, cellviability is an important readout as it determines the feasibility ofperforming critical downstream assays on CTCs, such as geneticprofiling. Since high flow rates can induce high shear rates which canbe detrimental to cell health, the cell viability was assessed in theOncoBean platform using the Invitrogen Live/Dead Assay. Briefly, H1650cells were spiked into serum free medium and processed throughanti-EpCAM coated OncoBean Chip at a flow rate of 10 mL/hr. Live/Deadreagent was flushed through the device and incubated for 15 minutes,followed by microscopic imaging of several 10× magnification fields ofview. The first four fields of view were considered as accurateestimates of cell viability. Live and dead cells were manually countedand cell viability was defined as the percent ratio of number of cellsalive to the total number of cells in the field of view. Using thisassay, 92.91±1.63% (mean±s.d.) of the cells were found to be viableafter flow at high throughput, comparable to the mean viability of 98.5%reported for the CTC-chip. These data indicate that despite facing aninitial momentary high stress at higher flow rates, cell viability isnot diminished compared to lower flow rates, which may be due to thecontinuous drop in shear stress that is produced by the radial flow.

Capture Profile:

The varying shear stress model proposed in the OncoBean Chip suggeststhere are differential regions of capture on the OncoBean, dependent onantigen density. For a given flow rate, capture of a high EpCAMexpressing cell would require less residence time for binding than a lowEpCAM expressing cell. This translates to distance traversed within thedevice, or the number of antibody-coated posts encountered by the cell.At low flow rates, the cells would be captured within a small distanceinto the device due to the radial drop in shear in addition to thealready-low shear rates. At high flow rates, a larger capture distancemay be required as the velocity should slow down for the cells to bind.To test this hypothesis, H1650 cells were spiked into blood and capturedat 1 mL/hr and 10 mL/hr. The cells captured were manually counted at1000 μm radial intervals. It was observed that with H1650, a cell linewith high EpCAM expression (greater than 500,000 antigens per cell),most cells are captured within the first half radius of the device at 1mL/hr, while the capture is more widespread at 10 mL/hr, utilizing alarger area of the device at the higher flow rate.

CTC Capture in Cancer Patients:

The strength of the Oncobean Chip was assessed by testing clinicalspecimens from different cancers. Whole blood from healthy donors (n=4)was processed as controls at 10 mL/hr to test for the specificity ofcapture against anti-EpCAM. After fixation of the cells on the device,the cells were permeabilized and stained for anti-Cytokeratin (CK)(tumor specific epithelial marker), anti-CD45 (leukocyte marker) andDAPI (nuclear stain) and respective secondary antibodies forimmunofluorescent imaging. FIGS. 41, 43A-D, and 44A-D presentrepresentative images of immunofluorescence staining of H1650 lungcancer cells along with WBCs captured on the chip. The criteria for CTCenumeration in patient samples was a CK+, CD45− and DAPI+ expressionprofile. The healthy controls gave a recovery of 1.4±0.89 CTCs per mL(range 0.91-2.67 CTCs per mL, median 1.13 CTCs per mL) as per the aboveenumeration criteria. Following this, a threshold of 2 CTCs per mL wasset for CTC detection in clinical specimens. Whole blood from pancreatic(n=2), breast (n=2) and lung (n=2) cancer patients were processedthrough the OncoBean Chip at 1 and 10 mL/hr in equal volumes to observeCTC recovery using an anti-EpCAM antibody. According to this criteria,CTCs were detected by the device in 100% of the samples, with a recoveryof 4.33±0.67 CTCs per mL at 1 mL/hr (range 3.33-5 CTCs per mL, median4.34 CTCs per mL) and 4.45±0.81 CTCs per mL at 10 mL/hr (range 3-5 CTCsper mL, median 4.84 CTCs per mL). The high flow rate of 10 mL/hr gaveequivalent recovery in 5 of 6 samples. Because of the small cohort ofspecimens tested, statistical significance was not observed. The CTCyields across the different cancers at the two flow rates are shown inFIG. 42. It can be seen that the high flow rate has a similar CTCrecovery capacity to that at a low flow rate in the various cancerspecimens processed.

DISCUSSION AND CONCLUSION

High throughput analysis has remained a hurdle in the retrieval ofcirculating tumor cells from cancer patients and is an issue in thedevelopment of new microfluidic technologies to measure CTCs. While manyphysical separation methods and integrated systems have been developed,the OncoBean Chip is an immuno-enrichment technique offering thespecificity of antibody-based capture in a high throughput manner (10mL/hr). The Oncobean Chip works by isolating CTCs from whole blood in asingle step, without the need for pre-processing or dilutions. TheOncobean Chip also achieves its high throughput functionality through asimple technique of a radial flow channel(s). Fluid simulations andtheoretical calculations show that the fluid velocity in the radial flowOncoBean Chip varies as 1/(2π rh), where r and h stand for the radiusand height of the channels, respectively. While increasing the height ofthe channel is an alternative to achieving high throughput,microfabrication of a high aspect ratio channel might be challenging. Aflow profile which dynamically varies with distance thus offers aneconomical and compact technology that enables the processing of highvolumes of fluid samples, which is highly advantageous in the context ofrare circulating tumor cells. The design of the OncoBean Chip can beoptimal for this purpose, as the Oncobean Chip achieves an averagecapture efficiency (>80%) comparable to that of the CTC Chip, whichstill remains a benchmark for microfluidic CTC extraction byimmuno-affinity methods Immuno-affinity based microfluidic platformslike the CTC Chip and others tend to perform poorly at high flow ratesdue to the linear flow profile wherein many cells either escape thesubstrate interactions or are travelling at a constantly high velocitythroughout the device in order for binding to occur. The OncoBean Chipshows stable binding at high flow rates due to the drop in velocity atevery radial position toward the outlets. Reducing shear at differentcross sections in the chip also allows a high flow rate withoutcompromising cell viability, increasing the feasibility of downstreamprocessing of these cells to obtain molecular characteristics andgenetic information. While high volumes of blood used for CTC analysispotentially offer higher yields of these rare cells, the detectionmethods also face the issue of an increased background contaminationowing to the quantity of sample. Further, WBC contamination tends not tobe a significant concern in the OncoBean Chip, as the high velocitiesprevented high nonspecific binding. There is a greater than 2-fold dropin the contaminating WBCs when run at 10 mL/hr in contrast to 1 mL/hr,and also a continuous drop in nonspecific binding with increasing flowrates. This is expected as the higher velocities reduce the interactiontime of these non-specific cells with the tumor-specific-antibody coatedposts, thereby reducing contamination. The higher levels of puritycoupled with higher CTC yields in a one-step strategy should enhanceaccuracy in molecular analyses of the recovered CTC population.

Affinity-based microfluidics depend, at least in part, onantibodyantigen kinetics, and antigen density is an important parametercontrolling the rate of capture. Typically, a cell with high EpCAMexpression such as H1650 would be captured early in the device as theantigen density would enhance bond formation. In contrast, a cell withlower EpCAM expression would be concentrated around the latter half ofthe device, nearer to the outlets, as the cells would need to be at asufficiently lower velocity for binding to occur due to the smallernumber of binding sites. The OncoBean Chip is thus conducive tocapturing cells with a wide range of EpCAM antigen expression. It isalso possible that multiple antibody combinations could be used in theOncoBean Chip to further fine tune CTC capture. The OncoBean Chip mayalso be used for clinical samples, capturing CTCs from the blood ofpatients with epithelial cancers by the use of anti-EpCAM as the captureantibody. Out of the 6 patient samples tested, 5 showed equivalentcapture at 10 mL/hr compared to capture at 1 mL/hr. This demonstratesthe potential of this technology as a high throughput platform toevaluate CTCs in different cancers. The technology also enables theprocessing of 7.5 mL of blood in under an hour, which is attractive fromthe perspective of a clinician. Accordingly, the OncoBean Chip is apotential tool for blood based diagnostics that can quickly become partof routine clinical tests.

The healthy controls also indicate the specificity of capture by theOncobean Chip. Although the small cohort of patients and the volume ofblood processed for unbiased comparison with that at low flow raterestrict a rigorous sensitivity analysis of the device, the captureefficiency of the device shows that the device may be used for highervolumes of blood for recovery of higher CTC numbers. Also,immunomagnetic technologies tend to suffer from limitations such asreduction in the binding affinities of antibodies on magnetic particles,requiring higher amounts of antibodies for larger cell populations.Tumor heterogeneity brings forth another set of challenges for physicalseparations as CTCs are observed to be widely different, with sizeheterogeneity being the most evident. While many such devices perform atcommendable throughputs, such size variations may result in significantCTC loss. The Oncobean Chip shows that high-throughput processing withimmuno-affinity capture is possible even without multiplexing such as inphysical separations which in many cases involve complex handling toreduce the blood volume.

The efficacy of the Oncobean Chip with cancer cell lines has beenvalidated and also with clinical specimens. In addition, it has alsobeen shown that the ultra-high throughput used is not detrimental to thecell viability and yields good purity and can be capitalized fordownstream studies. Further, the OncoBean Chip utilizes radial flow,which can be easily incorporated into other microfluidic chips ordevices for CTC culture. The captured cells can be released by widelyused cell recovery techniques or by incorporating thin coating ofhydrogels. The OncoBean Chip would be useful in the cases of early stagecancers where there may not be sufficient number of cells in thecirculation in order to be detected in 1-3 mL of blood, and accordinglyshows great promise as an early diagnostic tool.

Experimental Example Utilizing the OncoBean Chip:

COMSOL Simulations:

Finite element simulations were performed in COMSOL Multiphysics 4.2(Comsol Inc.) with an inlet flow rate of 10 mL/hr on a 6 mm radialsection and 30° arc of the proposed device. Navier-Stokes equations forincompressible fluid flow were used for the study. A symmetry boundarycondition was applied on the two similar boundaries flanking the posts.Wall (no slip) boundary condition was applied on the post outlines. Theparticle tracing plot was simulated with rigid particles 15 μm in size,with a condition of sticking to any encountered wall being applied.

Device Fabrication:

The design was prepared using AutoCAD software with the followingdimensions: bean width 50 μm, arc angles 90°, adjacent (lateral) postspacing 25-32 μm. The design was converted to a photomask (FineLineImaging) and used to prepare a mold by traditional photolithography.Briefly, a negative photoresist SU-8 100 (MicroChem Corp) was spincoated onto a silicon wafer at 2350 rpm. This was followed by softbaking at 65° C. for 10 min and 95° C. for 70 min, and then UV exposureof the pattern onto the wafer for 15 sec. Post exposure baking was doneat 65° C. for 3 min and 95° C. for 10 min and the pattern was developedin SU-8 developer. The wafer was then hard baked at 150° C. for 3 min. Apost height of 100 μm was achieved. Polydimethylsiloxane or PDMS(Ellsworth Adhesives) was prepared in a monomer to curing agent ratio of10:1 and baked overnight after degassing. The PDMS was peeled from themaster mold, cut and prepared for surface modification. Each PDMS chipwas bonded onto a glass slide using plasma bonding.3-mercaptopropylmethyldimethoxy silane (Gelest) was infused andincubated for 1 hour. This was followed by washing with ethanol andaddition of N-gamma-Maleimidobutyryloxy-Succinimide (GMBS)(ThermoScientifi c), a cross linking agent for 30 mins. The devices werewashed again and Neutravidin (Invitrogen-Life Technologies Inc.) wasadded and the devices were stored at 4° C. Before experiments, thedevices were incubated with biotin-conjugated anti-EpCAM (RnD Systems).

Cell Preparation:

Human lung cancer cell line H1650 and human breast cancer cell line MCF7were cultured in RPMI-1640 and DMEM (Invitrogen—LifeTechnologies, Inc.)respectively. The media were supplemented with 10% fetal bovine serum(Invitrogen—LifeTechnologies, Inc.). Both additionally contained 1%antibiotic-antimycotic solution. Cells were grown at 37° C. and 5% CO₂and medium was renewed every 2-3 days. Cells were passaged with 0.05%Trypsin-0.53 mM EDTA (Invitrogen—LifeTechnologies, Inc.).

Cell Capture and Analysis:

The cells were harvested with 0.05% Trypsin-0.53 mM EDTA and labeledwith CellTracker Green fluorescent dye (Invitrogen—LifeTechnologies,Inc.). They were counted with a hemocytometer and spiked into healthyblood or serum free medium. Informed consent was obtained from allhealthy blood donors at University of Michigan, Ann Arbor, USA using IRBapproved protocols. The devices were incubated with anti-EpCAM prior toexperiments. After antibody immobilization and wash, low dead volumetubing from Cole-Parmer (AAD02091-CP) was connected to the device andflow was facilitated through a syringe needle. 3% bovine serum albumin(Sigma Aldrich) was used as a blocking agent to reduce non-specificbinding. Two of the three outlets in the device were connected by ashort tubing to have a single outlet port for waste blood collection,while also increasing the fluid resistance. The fluid spiked with knownnumber of cells was then processed through the device at the respectiveflow rates. This was followed by washing with phosphate buffer saline(PBS) and fixing and permeabilization with BD Cytofi x/Cytoperm (BDBiosciences). DAPI (4′,6-diamidino-2-phenylindole)(Invitrogen—LifeTechnologies, Inc.) was then applied to stain thenucleus followed by a last washing step. The devices were stored at 4°C. until visualization with Nikon Eclipse Ti fluorescence microscope.Statistical analysis was performed with the software OriginPro 9.0. Astandard two-sample t-test was used for comparison between the groups.

Patient CTC Analysis: Informed consent was obtained from all donors.Whole blood from cancer patients was processed through the OncoBean Chipat the designated flow rates in equal volumes, followed by washing withphosphate buffer saline. The cells were fixed with 4% paraformaldehydeand refrigerated at 4° C. until immunofluorescent staining. Beforestaining, the cells were permeabilized with 0.2% Triton-X 100, followedby blocking with 2% goat serum in 3% bovine serum albumin Primaryantibodies anti-Cytokeratin 7/8 (BD Biosciences) and anti-CD45 (BDBiosciences) were applied in 1% bovine serum albumin for all sampleswith the exception of pancreatic cancer specimens where anti-Cytokeratin19 (SantaCruz Biotechnology Inc.) and anti-CD45 (SantaCruz BiotechnologyInc.) were used. After a quick wash, secondary antibodies AlexaFluor 488and AlexaFluor 546 or 568 (Invitrogen-Life Technologies Inc.) wereapplied in 1% bovine serum albumin DAPI was applied as the final stepbefore microscopic imaging.

Cell Viability Assay:

H1650 cells were harvested and spiked into serum free RPMI 1640 andprocessed through the OncoBean Chip at 10 mL/hr. The live/dead reagentconsisting of calcein AM and ethidium homodimer-1(Invitrogen—LifeTechnologies, Inc.) was prepared as specified bymanufacturer and applied to the cells in the device. Following 15 min ofincubation, several fields of view were microscopically imaged under 10×magnification.

One or more of the values described above may vary by ±5%, ±10%, ±15%,±20%, ±25%, etc. so long as the variance remains within the scope of thedisclosure. Unexpected results may be obtained from each member of aMarkush group independent from all other members. Each member may berelied upon individually and or in combination and provides adequatesupport for specific embodiments within the scope of the appendedclaims. The subject matter of all combinations of independent anddependent claims, both singly and multiply dependent, is hereinexpressly contemplated. The disclosure is illustrative including wordsof description rather than of limitation. Many modifications andvariations of the present disclosure are possible in light of the aboveteachings, and the disclosure may be practiced otherwise than asspecifically described herein.

What is claimed is:
 1. A system for capturing cells in a fluid, said system comprising: a substrate having a center and an outer edge and said substrate defining an axis at said center of said substrate, a fluid inlet at said axis, and a fluid outlet at said outer edge; a plurality of extensions extending outwardly from said substrate with each extension including a concave side having an arc facing toward said axis and said plurality of extensions arranged around said fluid inlet in a plurality of rows concentric with said fluid inlet with each one of said plurality of rows having an annular configuration, and said plurality of rows spaced from one another to define a curved channel between said plurality of rows and said plurality of extensions spaced from one another for enabling the fluid to move from said fluid inlet at said axis toward said fluid outlet at said outer edge of said substrate; and a functionalized graphene oxide disposed on each of said plurality of extensions with said functionalized graphene oxide interacting with the cells to immobilize the cells on said plurality of extensions when the cells come into contact with said functionalized graphene oxide.
 2. The system as set forth in claim 1 wherein said arc has an arc angle of from 75 to 90 degrees.
 3. The system as set forth in claim 1 wherein said channel is further defined between adjacent extensions of each of said plurality of rows with each extension in each of the plurality of rows configured to intercept movement of the fluid from said fluid inlet at said axis toward said fluid outlet at said outer edge of said substrate.
 4. The system as set forth in claim 3 wherein each one of said plurality of extensions is spaced from an adjacent one of said plurality of extensions a distance of from 10 to 100 μm.
 5. The system as set forth in claim 1 wherein each of said plurality of extensions includes gold.
 6. The system as set forth in claim 1 wherein said graphene oxide is functionalized with phospholipid-polyethylene-glyco-amine (PL-PEG-NH₂).
 7. The system as set forth in claim 1 wherein said graphene oxide is functionalized with the reaction product of phospholipid-polyethylene-glyco-amine (PL-PEG-NH₂) and N-γ-maleimidobutyryloxy succinimide ester (GMBS).
 8. The system as set forth in claim 7 wherein the reaction product is bonded to a protein.
 9. The system as set forth in claim 8 wherein said protein is bonded to an antibody for interaction with the cells.
 10. The system as set forth in claim 1 wherein each of said plurality of extensions has a height of 100 μm.
 11. The system as set forth in claim 1 wherein said system is a microfluidic device.
 12. The system as set forth in claim 1 wherein said channel has a height of from 1 to 1000 μm.
 13. The system as set forth in claim 1 wherein each extension has first and second ends and said arc has a center, and each extension has a first width of from 100 to 250 μm extending between said center of said arc and said first end and a second width of from 100 to 250 μm extending between said center of said arc and said second end.
 14. A microfluidic device for capturing cells in a fluid, said microfluidic device comprising: a silicon substrate having a center and an outer edge and said silicon substrate defining an axis at said center of said silicon substrate, a fluid inlet at said axis, and a fluid outlet at said outer edge; a plurality of metal extensions extending outwardly from said silicon substrate with each extension including a concave side having an arc facing toward said axis and said plurality of extensions arranged around said fluid inlet in a plurality of rows concentric with said fluid inlet with each one of said plurality of rows having an annular configuration, and said plurality of rows spaced from one another to define a curved channel between said plurality of rows and said plurality of metal extensions spaced from one another for enabling the fluid to move from said fluid inlet at said axis toward said fluid outlet at said outer edge of said silicon substrate; and a functionalized graphene oxide disposed on each of said plurality of metal extensions with said functionalized graphene oxide interacting with the cells to immobilize the cells on said plurality of extensions when the cells come into contact with said functionalized graphene oxide.
 15. The microfluidic device as set forth in claim 14 wherein said arc has an arc angle of from 75 to 90 degrees.
 16. The microfluidic device as set forth in claim 14 wherein said channel is further defined between adjacent metal extensions of each of said plurality of rows with each metal extension in each of the plurality of rows configured to intercept movement of the fluid from said fluid inlet at said axis toward said fluid outlet at said outer edge of said silicon substrate.
 17. The microfluidic device as set forth in claim 14 wherein each of said plurality of extensions includes gold.
 18. The microfluidic device as set forth in claim 14 wherein said graphene oxide is functionalized with phospholipid-polyethylene-glyco-amine (PL-PEG-NH₂).
 19. The microfluidic device as set forth in claim 14 wherein said graphene oxide is functionalized with the reaction product of phospholipid-polyethylene-glyco-amine (PL-PEG-NH₂) and N-γ-maleimidobutyryloxy succinimide ester (GMBS).
 20. The microfluidic device as set forth in claim 19 wherein the reaction product is bonded to a protein.
 21. The microfluidic device as set forth in claim 20 wherein said protein is bonded to an antibody for interaction with the cells.
 22. The microfluidic device as set forth in claim 14 wherein each of said plurality of metal extensions has a height of 100 μm.
 23. The microfluidic device as set forth in claim 22 wherein each of said plurality of metal extensions is spaced from an adjacent one of said plurality of metal extensions a distance of from 10 to 100 μm.
 24. The microfluidic device as set forth in claim 14 wherein said channel has a height of from 1 to 1000 μm.
 25. The microfluidic device as set forth in claim 14 wherein each extension has first and second ends and said arc has a center, and each extension has a first width of from 100 to 250 μm extending between said center of said arc and said first end and a second width of from 100 to 250 μm extending between said center of said arc and said second end.
 26. A method for capturing cells using a system comprising a substrate having a center and an outer edge and the substrate defining an axis at the center of the substrate, a fluid inlet at the axis, a fluid outlet at the outer edge, a plurality of extensions extending outwardly from the substrate with each extension including a concave side having an arc facing toward the axis and the plurality of extensions arranged around the fluid inlet in a plurality of rows concentric with the fluid inlet with each of the plurality rows having an annular configuration, and the plurality of rows spaced from one another to define a curved channel between the plurality of rows and the plurality of extensions spaced from one another, and a functionalized graphene oxide disposed on each of the plurality of extensions, said method comprising the steps of: introducing a sample of fluid containing the cells into the fluid inlet of the system such that the sample of fluid flows radially from the fluid inlet toward the fluid outlet at the outer edge of the substrate; and capturing the cells as the cells interact with the functionalized graphene oxide disposed on the plurality of extensions.
 27. The method as set forth in claim 26 wherein the step of introducing the sample of fluid is accomplished at a rate of up to about 10 mL/hr.
 28. The method as set forth in claim 27 wherein the rate decreases as the fluid flows from the inlet of the system towards the outer edge of the substrate. 